Antigenic combinations against francisella bacteria and related nanolipoprotein particles, compositions, methods and systems

ABSTRACT

Provided herein are antigenic combinations and related compositions, methods and systems for immunizing a host from an infection caused by Francisella bacterium. The antigenic combination comprises an antigenic polysaccharide component from a Francisella bacterium capable of triggering a humoral immune response in an individual, a protein antigen component from the Francisella bacterium capable of triggering a cellular immune response in the individual, and an adjuvant, the antigenic Francisella polysaccharide component, the Francisella protein antigen component and the adjuvant are in a suitable amount to immunize an individual against the Francisella bacterium.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application of U.S. application Ser. No. 16/422,263 filed on May 24, 2019, which in turn, claims priority to U.S. Provisional Application No. 62/676,222, entitled “Antigenic Combinations against Francisella bacteria and related Nanolipoprotein Particles, Compositions, Methods and Systems” filed on May 24, 2018 with docket number IL-13256, the content of each of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT GRANT

The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC for the operation of Lawrence Livermore National Laboratory.

TECHNICAL FIELD

The present disclosure relates to antigenic combinations for immunization against pathogenic Francisella bacteria and related nanolipoprotein particles, composition methods and systems.

BACKGROUND

Over the last few decades, there has been a constant drive towards developing a vaccine against Francisella species and in particular against Francisella tularensis. Despite the efforts, development of immunogenic compositions against bacterial infection caused by Francisella, bacteria and in particular of protective, stable and safe vaccines against more virulent Francisella species, is still challenging.

SUMMARY

Provided herein are antigenic combinations, and related nanolipoprotein particles, compositions, methods and systems which can be used to immunize an individual against a Francisella bacterium, which in several embodiments can provide improved immunostimulation and immunogenic response with respect to existing immunization methods.

According to a first aspect, an antigenic combination is described. The antigenic combination comprises an antigenic polysaccharide component from a Francisella bacterium capable of triggering a humoral immune response in an individual, a protein antigen component from the Francisella bacterium capable of triggering a cellular immune response in the individual, and an adjuvant. The antigenic Francisella polysaccharide component, the Francisella protein antigen component and the adjuvant are in a suitable amount to immunize an individual against the Francisella bacterium.

According to a second aspect, an immunogenic nanolipoprotein particle (NLP) is described, which presents the antigenic combination herein described. The immunogenic nanolipoprotein particle comprises: a scaffold protein, a membrane forming lipid, and the antigenic combination herein described, wherein the membrane forming lipids are arranged in a discoidal membrane lipid bilayer stabilized by the scaffold protein and the antigenic Francisella polysaccharide component, the Francisella protein antigen component, and the adjuvant are attached to the discoidal membrane lipid bilayer.

In some embodiments, at least one of a Francisella antigenic polysaccharide or derivative thereof of the antigenic Francisella polysaccharide component, a protein antigen from Francisella or a derivative thereof of the Francisella protein antigen component, and the adjuvant, comprises a hydrophobic region and is attached to the membrane lipid bilayer through interaction of the hydrophobic region with the membrane lipid bilayer.

In some embodiments, the immunogenic nanolipoprotein particle further comprises a functionalized membrane-forming lipid presenting an anchor compound substrate, and at least one of the antigenic polysaccharide from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium and the adjuvant, comprises an anchor compound attached to the anchor compound substrate of the functionalized amphipathic compound.

According to third aspect, an immunogenic composition is described. The immunogenic composition comprises the antigenic combination herein described together with a suitable vehicle, the antigenic combination in an amount effective to induce an immunogenic response against the Francisella bacterium in an individual. In some embodiments, in the composition the components of the antigenic combination are presented on a single same or different carrier (e.g. the composition comprises antigenic polysaccharide component, protein antigen component and adjuvant on a single NLP and/or antigenic polysaccharide component, protein antigen component and adjuvant on a microsphere). In some embodiments, in the composition the components of the antigenic combination are presented on separate same or different carriers (e.g. the composition comprises antigenic polysaccharide component on an NLP, the protein antigen component on a liposome and the adjuvant on a microsphere).

According to a fourth aspect, a method to provide an immunogenic nanolipoprotein particle is described. The method comprises: providing a membrane-forming lipid, a scaffold protein, the antigenic Francisella polysaccharide component, the Francisella protein antigen component, the adjuvant, and optionally a functionalized membrane-forming lipid presenting an anchor compound substrate

In the method, an antigenic polysaccharide from the Francisella bacterium or derivative thereof of the antigenic Francisella polysaccharide component, a protein antigen from the Francisella bacterium or a derivative thereof of the Francisella protein antigen component, and the adjuvant comprise a hydrophobic region configured for attachment to a membrane lipid bilayer, and/or comprise an anchor compound presented for binding with the anchor compound substrate on the functionalized amphipathic compound.

The method comprises mixing the membrane-forming lipid, the scaffold protein, the functionalized membrane-forming lipid and optionally the functionalized amphipathic compound, together with the antigenic polysaccharide from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium, and the adjuvant comprising a hydrophobic region, if any.

In the method the mixing is performed for a time and under conditions to allow self-assembly of the membrane-forming lipid, the scaffold protein, into a discoidal membrane lipid bilayer stabilized by the scaffold protein with the at least one of the Francisella antigenic polysaccharide or derivative thereof, the protein antigen from Francisella or derivative thereof and the adjuvant, if any, attached to the membrane lipid bilayer through interaction of the hydrophobic region with the membrane lipid bilayer to form a nanolipoprotein particle.

In embodiments wherein at least one of the antigenic polysaccharide from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium or derivative thereof, and the adjuvant comprise an anchor compound presented for binding with the anchor compound substrate on the functionalized amphipathic compound, the method further comprises mixing a formed nanolipoprotein particle comprising a functionalized amphipathic compound presenting an anchor compound substrate with the at least one of the antigenic polysaccharide or derivative thereof, the protein antigen and the adjuvant for a time and under condition to allow the attaching to the nanolipoprotein particle through the binding of the anchor compound and the anchor compound substrate of the functionalized membrane-forming lipid.

According to a fifth aspect, a method to provide an immunogenic composition is described. The method comprises mixing an antigenic polysaccharide component from a Francisella bacterium, at least one protein antigen component from the Francisella bacterium and an adjuvant together with a suitable vehicle, the antigenic polysaccharide component, the protein antigen component, and the adjuvant in a suitable form and amount to immunize an individual against the Francisella bacterium.

According to a sixth aspect, a method of immunizing an individual against an infection of a Francisella bacterium is described. The method comprises administering to the individual an effective amount of the antigenic combination, the immunogenic nanoparticle and/or immunogenic composition described herein comprising an antigenic polysaccharide component from the Francisella bacterium, and a protein antigen component from the Francisella bacterium in a suitable form and amount to immunize an individual against the Francisella bacterium.

According to a seventh aspect, a system for immunizing an individual against an infection of a Francisella bacterium is described. The system comprises an antigenic combination herein described wherein the antigenic Francisella polysaccharide component, the Francisella protein antigen component are from the Francisella bacterium. In the system the antigenic polysaccharide component and the protein antigen component from the Francisella bacterium are comprised in separate compositions in a form and an amount for simultaneous, combined or sequential use in the method to immunize an individual against the Francisella bacterium described herein.

In antigenic combinations, and related nanolipoprotein particles, compositions, method and systems herein described, the antigenic Francisella polysaccharide component comprises an antigenic Francisella and/or a derivative thereof, and the antigenic Francisella protein antigen component comprises an antigenic Francisella protein antigen and/or a derivative thereof.

The antigenic combinations, and related nanolipoprotein particles, compositions, method and systems herein described allow in several embodiments to effectively immunize an individual as a result of a synergistic effect in protection against Francisella bacteria and in particular against bacteria of the species Francisella tularensis subspecies tularensis, particularly type A Francisella tularensis.

The antigenic combinations, and related nanolipoprotein particles, compositions, method and systems herein described allow in several embodiments induction of both humoral and cell mediated immunity in an immunized individual, thus leading to enhanced protective capacity against a virulent Francisella bacterium and in particular against virulent bacteria of the species Francisella tularensis over either component alone.

In particular, when the antigenic combinations are presented on a single carrier and in particular on an immunogenic nanolipoprotein particle, the co-localized presentation of the antigens results in an improved immunostimulation and immunogenic response with respect to known immunogenic compositions and other immunogenic compositions herein described.

The antigenic combinations, and related nanolipoprotein particles, compositions, method and systems herein described allow in several embodiments development of a safer vaccine with an effective protection against infections caused by Francisella tularensis.

The antigenic combinations, and related nanolipoprotein particles, compositions, method and systems herein described can be used in connection with various applications wherein use of antigens of a Francisella bacterium, and/or immunization of an individual against a Francisella bacterium are desired. For example, antigenic combinations, and related nanolipoprotein particles, compositions, method and systems herein described can be used in combination with compounds such as therapeutics to target Francisella bacterium in an individual, and in particular in combination with immunostimulating agents against infection of the individual by a Francisella bacterium, in vaccine development and use, and/or to treat and/or prevent a condition associated with an infection of a Francisella bacterium, such as Francisella tularensis. Additional exemplary applications include basic biology research, applied biology, bio-engineering, medical research, medical diagnostics, therapeutics, and additional fields identifiable by a skilled person upon reading of the present disclosure.

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and example sections, serve to explain the principles and implementations of the disclosure.

FIG. 1 shows a schematic illustrating the general chemical structure of bacterial lipopolysaccharides comprising Lipid A and a polysaccharide component. Different components of the bacterial lipopolysaccharides are indicated with symbols shown in different shades of gray as indicate in the figure.

FIGS. 2A-C show a schematic representation of an NLP assembly process to provide an NLP comprising a functionalized membrane-forming lipid presenting at least one protein antigen according to some embodiments herein described. In the illustration of FIGS. 2A-C, the starting components, intermediate NLP and final NLP presenting an anchor bearing immunological agents are illustrated in FIGS. 2A to 2C respectively.

FIG. 3 shows a schematic representation of an NLP assembly with the incorporation of an adjuvant, a LPS and a protein antigen both from Francisella tularensis.

FIG. 4 illustrates exemplary embodiments of the immunogenic agents herein described loaded in a nanolipoprotein particle carrier.

FIGS. 5A-E illustrate exemplary structures of Francisella LPS and its components from Okan 2013 [1]. In particular, FIG. 5A shows a representative structure of Francisella LPS having a major Francisella lipid A molecule with a polysaccharide core component. Chain length variation (16/18) indicates temperature regulation. FIG. 5B shows less abundant lipid variants having various modifications in sugar composition as shown in dashed circles. FIGS. 5C and 5D show the structure of F. tularensis O-antigen and F. novicida O-antigen, respectively. FIG. 5E shows the structure of E. coli Kdo2-lipid A.

FIG. 6 illustrates an exemplary embodiment of F. tularensis LVS lipid A from LVS F. tularensis from Okan 2013 [1].

FIG. 7 illustrates a cartoon representation of Francisella LPS and its modifying proteins.

FIGS. 8A-H show in some embodiments size exclusion chromatography analysis of various NLP assemblies with or without LPS at different temperatures. As monomeric LPS species should incorporate more readily into NLPs than large aggregates, conditions were screened to dissociate large LPS aggregates typically found in aqueous preparations of LPS. Cholate (60 mM) (left column) or Z3-14 (60 mM) (right column) were first added to LPS stock (LPS rehydrated in water) and incubated for about 2 minutes at elevated temperatures prior to mixing with NLP assembly reaction components. Three incubation temperatures were tested: 37° C., 55° C. and 95° C. After thorough dialysis to remove residual cholate, the assembly was analyzed by size exclusion chromatography. The results show that incubating LPS in detergent at 95° C. results in NLP formation. As a control, NLP formation in the absence is shown in the top two chromatograms, using either cholate (left column) or the surfactant Z3-14 (right column) to solubilize lipidic components of the reaction into aqueous solution.

FIGS. 9A-D show in some embodiments size exclusion chromatography analysis of various NLP assemblies prepared with LPS that has been incubated in cholate at 95° C. for various times. Cholate at 60 mM was added to LPS stock and incubated at 95° C. for different incubation times: 2 minutes, 5 minutes, and 15 minutes. The LPS stock mixed with cholate was then added to lipid, and then mixed with scaffold protein. After thorough dialysis to remove residual cholate, the assembly was analyzed by size exclusion chromatography. The results show that incubating LPS in cholate at 95° C. for 15 minutes was optimal for NLP formation.

FIG. 10 shows size exclusion chromatography analysis of three exemplary NLP assemblies with LPS and NLP at different ratios: no LPS (0 μg LPS/μg rE4, NP1080) (black solid trace), a low amount of LPS (0.29 μg LPS/μg rE4, NP1079) (gray solid trace), and a high amount of LPS (0.86 μg LPS/μg rE4, NP1078) (black dashed trace).

FIG. 11A shows representative RP-HPLC traces of pure F.t LVS LPS standards at different concentrations. LPS samples were diluted in water and injected onto the C8 RP column, using an aqueous buffer and two organic buffers (acetonitrile and isopropanol) to provide appropriate resolution. An evaporative light scattering detector was used to detect the LPS. The peak areas were calculated to prepare a standard curve for quantification of unknown samples. FIG. 11B shows a standard curve based on the peak area of known LPS standards. This standard curve enables the quantification of LPS in unknown samples by correlating LPS peak area values with LPS concentration.

FIG. 12 shows representative SEC traces of NiNLPs prepared with LPS (at three different amounts: no LPS, low LPS incorporation, high LPS incorporation, shown in FIG. 10) are able to bind his-tagged protein. His-tagged protein (black trace) is incubated with the NiNLP (gray trace) to form protein:NiNLP complex (dashed trace). The increased absorbance intensity of the protein:NiNLP complex, as well as the shift to earlier retention times, indicates that conjugation was successful. Protein conjugation was successfully demonstrated using NiNLP with no LPS, with low amounts of incorporated LPS, and high amounts of incorporated LPS. This demonstrates that the presence of LPS in the NLP does not interfere with the incorporation or conjugation of additional biomolecules.

FIG. 13A shows exemplary SEC traces of a scaled-up assembly of LPS:NiNLPs in two purification runs. Samples were purified on a Superose 6 SEC column (10mm×30cm column dimensions, 1 ml/min PBS flow rate). Both purification runs were identical with respect to chromatogram intensity and elution profile. 1 mL fractions were collected from 7 to 17 minutes for subsequent analysis. Appropriate fractions corresponding to NLPs were pooled for subsequent analysis. FIG. 13B shows exemplary SEC analysis of pre-concentrated and post-concentrated LPS:NiNLPs. Some material is lost during the purification status (as indicated by the decrease is chromatogram intensity post-concentrated), but the peak shape remains. This indicates that the NLP remain intact after the concentration process.

FIG. 14 shows exemplary SEC analysis of lyophilized and non-lyophilized NLPs samples. To verify that LPS:NiNLPs could be lyophilized while maintaining particle integrity, LPS:NiNLPs were formulated with trehalose and either stored at 4° C. in solution (control) or lyophilized. These samples were stored overnight. The lyophilized sample was subsequently rehydrated in water and analyzed by analytical SEC. LPS:NiNLPs stored at 4° C. in solution (black trace) and lyophilized, rehydrated LPS:NiNLPs (gray trace) exhibited identical peak shape and overall chromatogram intensity.

FIG. 15A shows exemplary SEC analysis of IglC and CpG conjugation to NiNLPs (in the absence of incorporated LPS) to form CpG:IglC:NiNLP formulations. IglC conjugates to NiNLPs (black trace) to form IglC:NiNLP (gray trace). Subsequent addition of cholesterol-tagged CpG forms CpG:IglC:NiNLP (dashed trace). FIG. 15B shows exemplary SEC analysis of IglC and CpG conjugation to LPS:NiNLPs (in the presence of incorporated LPS) to form CpG:IglC:LPS:NiNLP formulations. IglC conjugates to LPS:NiNLPs (black trace) to form IglC:LPS:NiNLP (gray trace). Subsequent addition of cholesterol-tagged CpG forms CpG:IglC:LPS:NiNLP (dashed trace). These tests demonstrate that vaccine formulations co-localizing LPS antigen, protein antigen, and adjuvant can be successfully prepared.

FIG. 16 shows in some embodiments SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of LPS, both unincorporated and incorporated in NiNLPs. For each sample, an equivalent amount of LPS (280 ng) was loaded on an SDS-polyacrylamide gel to compare band intensities and pattern. For Western analysis, samples were transferred to a membrane and probed with the anti-F.t. LPS antibody FB11, and imaged using a fluorescent secondary antibody. Marker corresponds to a protein standard marker included for general comparisons of molecular weights. The banding pattern is identical between the unincorporated LPS and the LPS incorporated in the NLP.

FIG. 17A shows an exemplary SEC of an LPS:NiNLP assembly. Fractions were purified on a Superose 6 SEC column (10mm×30cm column dimensions, 1mL/min PBS flow rate) collected every 1.3 minutes between 7 and 20 minutes. In FIG. 17B, these fractions are further analyzed by analytical SEC (Superose 6 column, 10mm×30cm, 0.2mL/min PBS flow rate) to demonstrate the relative size of the NLPs in each purified fraction. FIG. 17C shows exemplary SDS-PAGE analysis of SEC fractions. Fractions were either stained for protein constituents (i.e. apoE) using the fluorescent stain SyproRuby, or transferred to a membrane and probed by Western analysis with the anti-F.t. LPS antibody FB11. These gels demonstrate that fractions corresponding to NLPs (f2-f7) exhibit the presence of both LPS and apoE. In addition, the distribution of LPS molecules correlates with the size of the NLP. Larger NLP (collected in early fractions) are predominated by LPS species of high molecular weight, whereas smaller NLPs (collected in later fractions) are predominated by LPS species of lower molecular weight.

FIG. 18 illustrates in some embodiments percent survival of the tested animals after being challenged with Francisella tularensis aerosol.

FIGS. 19A-B illustrate in some embodiments the individual animal weight measured over the course of the experiments for experimental groups 1-3 (FIG. 19A) and group 4-5 (FIG. 19B).

FIG. 20 illustrates in some embodiments bacterial burden in cfu/g measured in lungs and spleens 21 days post challenge in two survival groups (groups 4 and 5).

FIGS. 21A-B show in an exemplary SDS-PAGE and Western blot analysis of the vaccine formulations used in the animal tests. Group 1: PBS vehicle only (intranasal vaccination); Group2: CpG:LPS:NiNLP (intranasal vaccination); Group 3: CpG:IglC:NiNLP (intranasal vaccination); Group 4: CpG:IglC:LPS:NiNLP (intramuscular vaccination); Group 5: CpG:IglC:LPS:NiNLP (intranasal vaccination); Group 6: CpG+IglC+LPS (intranasal vaccination). In FIG. 21A, vaccine formulations were analyzed by SDS-PAGE to verify presence of appropriate protein constituents in the formulations to verify the presence of NLP (i.e. apoE in Groups 2-5) and antigen (i.e. IglC in Groups 3-6). In FIG. 21B, SDS-PAGE and Western blot (using anti F.t. LVS LPS antibody FB11) were used to verify presence of F.t. LPS in the appropriate formulation groups (i.e. Groups 2, 4-6).

FIG. 22 illustrates in some embodiments percent survival of the tested animals after being challenged with Francisella tularensis aerosol. Group 1: PBS vehicle only (intranasal vaccination); Group2: CpG:LPS:NiNLP (intranasal vaccination); Group 3: CpG:IglC:NiNLP (intranasal vaccination); Group 4: CpG:IglC:LPS:NiNLP (intramuscular vaccination); Group 5: CpG:IglC:LPS:NiNLP (intranasal vaccination); Group 6: CpG+IglC+LPS (intranasal vaccination). This figure is taken from Sun et al. [2].

FIG. 23 illustrates in some embodiments the individual animal weight measured over the course of the experiments for experimental group 4 and group 6. Group 4: CpG:IglC:LPS:NiNLP (intramuscular vaccination); Group 6: CpG+IglC+LPS (intranasal vaccination).

FIGS. 24A-B show an exemplary graphical representation of the daily clinical scores recorded during the 21-day observation period after the Francisella tularensis aerosol challenge.

FIG. 25 illustrates a stereoview of the crystal structure of an exemplary Francisella protein antigen IglC. The 13 β-strands are numbered and the helices are labeled A-E (including α-helices A and C and 3₁₀-helices B, D, and E). The image is taken from Sun et al. [2].

FIG. 26 shows the search parameters and databases used for querying the protein sequences.

FIG. 27A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 2 as a query sequence. FIG. 27B is a grayscale representation of a graphic summary of the BLASTP search results using IglC protein having SEQ ID NO: 2 as a query sequence.

FIG. 28A shows the alignment hit table generated from the BLASTP search using SEQ ID NO: 4 as a query sequence. FIG. 28B is a grayscale representation of a graphic summary of the BLASTP search results using DnaK protein SEQ ID NO: 4 as a query sequence.

FIG. 29A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 6 as a query sequence. FIG. 29B is a grayscale representation of a graphic summary of the BLASTP search results using OmpA protein SEQ ID NO: 6 as a query sequence.

FIG. 30A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 8 as a query sequence. FIG. 30B is a grayscale representation of a graphic summary of the BLASTP search results using SucB protein SEQ ID NO: 8 as a query sequence.

FIG. 31A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 88 as a query sequence. FIG. 31B is a grayscale representation of a graphic summary of the BLASTP search results using FopA protein SEQ ID NO: 88 as a query sequence.

FIG. 32A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 10 as a query sequence. FIG. 32B is a grayscale representation of a graphic summary of the BLASTP search results using LpnA/Tul4 protein SEQ ID NO: 10 as a query sequence.

FIG. 33A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 14 as a query sequence. FIG. 33B is a grayscale representation of a graphic summary of the BLASTP search results using FTT1416c protein SEQ ID NO: 14 as a query sequence.

FIG. 34A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 16 as a query sequence. FIG. 34B is a grayscale representation of a graphic summary of the BLASTP search results using FTT0890c protein SEQ ID NO: 16 as a query sequence.

FIG. 35A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 18 as a query sequence. FIG. 35B is a grayscale representation of a graphic summary of the BLASTP search results using FopB protein SEQ ID NO: 18 as a query sequence.

FIG. 36A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 20 as a query sequence. FIG. 36B is a grayscale representation of a graphic summary of the BLASTP search results using FTT1676 protein SEQ ID NO: 20 as a query sequence.

FIG. 37A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 22 as a query sequence. FIG. 37B is a grayscale representation of a graphic summary of the BLASTP search results using FTT1441 protein SEQ ID NO: 22 as a query sequence.

FIG. 38A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 24 as a query sequence. FIG. 38B is a grayscale representation of a graphic summary of the BLASTP search results using FTT1778c protein SEQ ID NO: 24 as a query sequence.

FIG. 39A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 25 as a query sequence. FIG. 39B is a grayscale representation of a graphic summary of the BLASTP search results using FTT0814c protein SEQ ID NO: 25 as a query sequence.

FIG. 40A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 26 as a query sequence. FIG. 40B is a grayscale representation of a graphic summary of the BLASTP search results using FTT1043 protein SEQ ID NO: 26 as a query sequence.

FIG. 41A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 27 as a query sequence. FIG. 41B is a grayscale representation of a graphic summary of the BLASTP search results using FTT0438 protein SEQ ID NO: 27 as a query sequence.

FIG. 42A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 28 as a query sequence. FIG. 42B is a grayscale representation of a graphic summary of the BLASTP search results using FTT0071 protein SEQ ID NO: 28 as a query sequence.

FIG. 43A shows a representation of the alignment hit table generated from the BLASTP search using SEQ ID NO: 29 as a query sequence. FIG. 43B is a grayscale representation of a graphic summary of the BLASTP search results using FTT0289c protein SEQ ID NO: 29 as a query sequence.

FIG. 44 illustrates in a plot the IglC-specific immune responses in Fischer 344 rat splenocytes. This figure represents the results from an ELISPOT assay to identify peptides within the IglC protein that stimulate IFNγ production by antigen-specific T cells from Fischer 344 rats immunized with a recombinant Listeria monocytogenes strain engineered to express the Francisella tularensis IglC protein (Lm-IglC). One Fischer 344 rat was vaccinated IM with 1×10⁸ CFU of Lm-IglC and and then boosted IV 4 weeks later with 1×10⁸ CFU Lm-IglC. The splenocytes were harvested seven days after the boost and restimulated the next day with individual peptides that span the length the IglC protein shown on the X-axis. The number of IFNγ specific spot forming cells (SFC) per 200,000 lymphocytes are shown on the Y axis. Specific reactivity is indicated by a peak in the number of IFNγ secreting cells. The results show that there are potentially 5 epitopes that stimulate T cells in Fischer 344 rats.

FIG. 45 illustrates in an exemplary embodiments flow cytometry analyses of T cells from IglC-immunized Fischer 344 rats to identify CD8 T cell epitopes. This figure shows that IglC peptide #9 is a CD8 T cell antigen in Fischer 344 rats. One Fischer 344 rat was vaccinated IV with 1 ×10⁸ CFU Lm-IglC and then boosted 2 weeks later with 1×10⁷ CFU IV Lm-IglC. Splenocytes were isolated six days after the boost and restimulated with pools of peptides from IglC (IglC pools 1+2) or listeriolysin O (LLO pool) or with individual IglC peptides #4, #9, #16, #38, and #44 as shown on the X-axis. The percent of CD8⁺ T cells expressing intracellular IFNγ is shown on the Y-axis. The results show that only IglC peptide 9 stimulated CD8 T cells to produce IFNγ, indicating that it is the only CD8 T cell epitope within the IglC protein.

FIG. 46 illustrates in a plot the mapping IglC-specific CD8+ T cell epitope in Fischer 344 rats. This figure represents the results from an IFNγ ELISPOT assay to identify the specific 9 amino acid peptide within the 15 amino acid IglC peptide #9 that stimulated IFNγ production by CD8 T cells from immunized Fischer 344 rats. Fischer 344 rats were vaccinated IV with 1×10⁸ CFU Lm-IglC and then boosted IV 6 weeks later with 1×10⁷ CFU Lm-IglC. Six days after the boost, splenocytes were harvested and restimulated with overlapping 9 amino acids peptides that span the 15 amino acid IglC peptide #9 as shown on the Y-axis. The X-axis represents the IFN-γ response, enumerated as the number of IFN-γ specific spot-forming cells (SFC) per 200,000 lymphocytes. The results show that the sequence of the stimulatory peptide is NCRLFIDSL.

DETAILED DESCRIPTION

Provided herein are antigenic combinations, and related nanolipoprotein particles, compositions, method and systems which can be used to immunize an individual against a Francisella bacterium, and in particular against a bacterium of the species Francisella tularensis.

The term “Francisella” as used herein indicates a genus of pathogenic, Gram-negative bacteria. Francisella is the sole genus in the family Francisellaceae, falling within the class Gammaproteobacteria. Francisella bacteria are small coccobacillary or rod-shaped, nonmotile organisms, which are also facultative intracellular parasites of macrophages. Strict aerobes, Francisella colonies bear a morphological resemblance to those of the genus Brucella. Francisella contains two recognized species, F. tularensis and F. philomiragia, several recently identified species F. noatunensis, F. hispaniensis, and possibly F. halioticida as well as potentially other as yet unidentified species, underscoring the dynamic nature of Francisella taxonomy [3-6]. Accordingly, exemplary bacteria of the genus Francisella are F. tularensis, F. novicida, F. hispaniensis, W. persica, F. noatunensis, F. philomiragia, F. halioticida, F. endociliophora, F. guangzhouensis, and F. piscicida.

The term “Francisella tularensis” “F. tularensis” or “FT' as used herein indicates a small, highly pleomorphic, gram-negative coccobacillus. In particular, Francisella tularensis indicates a pathogenic species of Gram-negative, rod-shaped coccobacillus, an aerobe bacterium. FT is a non-spore forming, non-motile bacteria and the causative agent of tularemia, the pneumonic form of which is often lethal without treatment. When found in nature, Francisella tularensis can survive for several weeks at low temperatures in animal carcasses, soil, and water. In laboratory, Francisella tularensis appears as small rods (0.2 by 0.2 μm), and is grown best at 35-37 degrees Celsius. F. tularensis contains four recognized subspecies: F. tularensis subspecies tularensis, F. tularensis subspecies holarctica, F. tularensis subspecies novicida and F. tularensis subspecies mediasiatica [7].

The term “antigen” or “antigenic” as used herein indicates a substance that prompts the generation of antibodies and/or can cause an immune response in an individual. In particular, antigens in the sense of the present disclosure encompass all substances that can be recognized by an adaptive immune system. Exemplary antigens include exogenous antigens and endogenous antigens. Exogenous antigens are antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. Exogenous antigens are taken into the antigen-presenting cells (APCs) by endocytosis or phagocytosis, and processed into fragments. APCs then present the fragments to T helper cells (CD4⁺) by the use of class II histocompatibility molecules on their surface. Some T cells are specific for the peptide: MHC complex. T cells become activated and start to secrete cytokines. Cytokines are substances that can activate cytotoxic T lymphocytes (CTL), antibody-secreting B cells, macrophages, and other particles. Endogenous antigens are antigens that have been generated within the cell, as a result of normal cell metabolism, or because of viral or intracellular bacterial infection. The fragments are then presented on the cell surface in the complex with MHC class I molecules. If activated cytotoxic CD8⁺ T cells recognize them, the T cells begin to secrete various toxins that cause the lysis or apoptosis of the infected cell. In order to keep the cytotoxic cells from killing cells just for presenting self-proteins, self-reactive T cells are deleted from the repertoire as a result of tolerance (also known as negative selection). They include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous) antigens. Antigens are also generated between normal cells.

In embodiments of the disclosure an antigenic combination described is configured to immunize an individual against a Francisella bacterium, and in particular against a Francisella tularensis bacterium.

The term “immunize” or “immunogenize” as used herein refers to the ability of a particular antigenic substance to provoke an immune response in an individual. In particular, an immune response indicates the reaction of the cells and fluids of the body of an individual to the presence of a substance that is not recognized as a constituent of the body itself. The immune response can be a humoral (antibody) response, or a cell-mediated response, or both.

The term “cell mediated response” in the sense of the disclosure indicates an immune response that does not involve antibodies, but rather involves the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. The protective function of cell mediated immunity is typically associated with cells, such as CD4 cells or helper T cells and Naive T cells. Mature T cells that have yet to encounter an antigen, are converted into activated effector T cells after encountering antigen-presenting cells (APCs). These APCs, such as macrophages, dendritic cells, and B cells in some circumstances, load antigenic peptides onto the MHC of the cell, in turn presenting the peptide to receptors on T cells. The most important of these APCs are highly specialized dendritic cells; conceivably operating solely to ingest and present antigens. Activated Effector T cells can be placed into three functioning classes, detecting peptide antigens originating from various types of pathogen: The first class being Cytotoxic T cells, which kill infected target cells by apoptosis without using cytokines, the second class being TH1 cells, which primarily function to activate macrophages, and the third class being TH2 cells, which primarily function to stimulate B cells into producing antibodies. Cellular immunity protects the body by: i) T-cell mediated immunity or T-cell immunity activating antigen-specific cytotoxic T cells that are able to induce apoptosis in body cells displaying epitopes of foreign antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; ii) activating macrophages and natural killer cells, enabling them to destroy pathogens; and iii) stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

Cell mediated immune responses triggered by an antigen can be detected using standard recall response assays using the antigen. The assays typically comprise harvesting spleens from vaccinated animals vaccinated with the antigen, processing the harvested spleen into single cell suspension and re-stimulating total splenocytes in the suspension with purified antigen for 24-72 hours. The assays further comprise collecting culture supernatants following re-stimulation and analyzing the collected culture supernatants for presence of TH1/Th2 cytokines such as IL-2 and IFN-γ to assess T cell responses. Alternatively, assays can comprise harvesting spleens from vaccinated mice, processing harvested spleens into a single cell suspension and purifying CD4+ and CD8+ T cells, e.g. using MACS bead sorting prior to re-stimulating the purified CD4+ and CD8+ T cells with purified antigen. The assays further comprise collecting culture supernatants and analyzing the collected culture supernatants for presence of IL-2 and IFN-γ to assess t cell responses. An additional assay that can be used to detect cell mediate immune response is the Enzyme-Linked ImmunoSpot (ELISPOT) assay is also widely used to monitor cell-mediated immune responses in animals.

The term “humoral immunity” in the sense of the disclosure indicates an immune response mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Humoral immunity is so named because it involves substances found in the humors, or body fluids. It contrasts with cell-mediated immunity. Its aspects involving antibodies are often called antibody-mediated immunity. Humoral immunity refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination. In a humoral immune response, first the B cells mature in the bone marrow and gain B-cell receptors (BCR's) which are displayed in large number on the cell surface. These membrane-bound protein complexes have antibodies which are specific for antigen detection. Each B cell has a unique antibody that binds with an antigen. The matured B cells migrate from bone marrow to lymph nodes or other lymphatic organs, where they begin to encounter pathogens. The humoral response is typically divided in B cell activation, B cell proliferation and Antibody-antigen reaction. B cell action refers to the phase where when a B cell encounters an antigen, it is bound to the receptor and taken inside by endocytosis. The antigen is processed and presented on its surface again with MHC-II molecule. Be cell proliferation refers to the phase where the B cell waits for the T_(H) cell to bind to the complex and this binding will activate T_(H) cell and it releases cytokines that induce B cells to divide rapidly which make thousands of identical clones of B cell. These daughter cells either become plasma cells or memory cells. The memory B cells remain inactive here; later when these memory B cells encounter the same antigen due to reinfection, they divide and form Plasma cells. On the other hand, the plasma cells produce a large number of antibodies which are released free in the circulatory system. The antibody antigen reaction refers to the phase where these antibodies will encounter antigens and bind with them. This will either interfere with the chemical interaction between host and foreign cells, or they may form bridges between their antigenic sites hindering their proper functioning, or their presence will attract macrophages or killer cells to phagocytose them.

A humoral response can be qualitatively or quantitatively detected with standard ELISAs methods and other methods identifiable by a person of ordinary skill in the art.

Antigenic molecules capable of triggering a cellular and/or humoral comprise one or more epitopes. The term “epitope” as used herein, also known as an “antigenic determinant” refers to the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. For example, the epitope is the specific piece of the antigen to which an antibody binds. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes.

Epitopes of antigenic molecules can be conformational epitopes or linear epitopes, based on their structure and interaction with the paratope as will be understood by a skilled person. A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence and interacts with a corresponding paratope based on the 3-D surface features and shape or tertiary structure of the antigen. Linear epitopes interact with a corresponding paratope based on their primary structure. A linear epitope is typically formed by a continuous sequence of residues of an antigen.

Epitopes can be mapped, for example using protein microarrays, or with ELISA or ELISPOT techniques, among others known to those skilled in the art. Another technique involves high-throughput mutagenesis, an epitope mapping strategy developed to improve rapid mapping of conformational epitopes on structurally complex proteins [8]. In addition, MHC class I and II epitopes can be predicted by computational means [9]. Additional methods for identifying epitopes are described in U.S. Pat. Nos. 8,889,142, 8,486,411, 7,754,228 and 6,635,746 and will be understood by a person skilled in the art.

In the embodiments herein described, the antigenic combination comprises an antigenic polysaccharide component from a Francisella bacterium capable of triggering a humoral immune response, a protein antigen component from Francisella bacterium capable of triggering a cell-mediated immune response, and an adjuvant.

In particular, in antigenic combination herein described the antigenic polysaccharide component can comprise an antigenic polysaccharide from a Francisella bacterium capable of triggering a humoral immune response and/or a derivative thereof, and a protein antigen from Francisella bacterium capable of triggering a cell-mediated immune response and/or a derivative thereof.

The term “derivative” as used herein in connection with an antigenic polysaccharide or a protein antigen, indicates a fragment of the molecule which retains the immunogenicity of the original antigenic polysaccharide or protein with a same lower or higher degree. Typically, a derivative of an antigenic molecule comprises one or more epitopes of the original antigen which form the base for obtaining the derivative. Derivatives can be obtained by fragmentation of a base antigenic polysaccharides or protein antigen, or can be provided synthetically to reproduce part of the structure of the base antigenic polysaccharide or protein antigen as will be understood by a skilled person.

In some embodiments, rationally designed antigenic fragments and synthetic polysaccharides or peptides comprising one or more epitopes which maintain the immunogenicity of the original antigenic polysaccharides or protein antigens can serve as the derivatives. For example, polynucleotides can be designed to encode a protein fragment to include one or more epitopes of a protein antigen. In some embodiments, polynucleotides can be designed to encode a mutated protein from the original protein antigen with a same, lower or higher degree of immunogenicity. In some embodiments, the polynucleotide can be designed such that the resulting protein, protein fragment or mutated proteins is expressed as a fusion, or chimeric protein products by joining via a peptide bond to a heterologous protein sequence of a different protein to facilitate purification or detection. For example, a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product. In particular, in some embodiments, the polynucleotide can be engineered so that the protein antigen is labeled or tagged. Labeling or tagging can be performed with methods that include, for example, FRET pairs, NETS-labeling, fluorescent dyes, and biotin as well as coding for a “His-tag” to enable protein isolation and purification via established Ni-affinity chromatography.

Derivatives of antigenic polysaccharides can be chemically synthesized through chemical polymerization as will be understood by a person skilled in the art. Examples of antigenic polysaccharide derivatives can comprise at least one tetrasaccharide repeating unit as described below.

Derivatives of original antigenic proteins and polysaccharides can be assessed for immunogenicity and protective capacity in a suitable animal model using approaches known to a person skilled in the art. Murine model systems can be employed where animals are vaccinated with the antigen of interest and the humoral and cell mediated immune response to vaccination is characterized. In the case of humoral responses, serum from vaccinated animals can be screened for the presence of antigen specific antibodies using traditional ELISA methodologies. Cell mediated immunity can be assessed using recall responses to quantify the ability of recombinant protein to elicit activation of T cells in vaccinated animals. Protection can then be assessed in vaccinated animals that are subsequently challenged with a lethal dose of Francisella and assessed for survival.

The term “polysaccharide” as used herein indicates a polymeric saccharide having different lengths and dimensions. In particular, polysaccharides in the sense of the disclosure encompass polymeric carbohydrate molecules composed of chains of monosaccharide units bound together by glycosidic linkages, which on hydrolysis give the constituent monosaccharides and/or oligosaccharides. A “glycosidic linkage” as used herein indicates an oxygen atom that joins an anomeric carbon atom of one monosaccharide to a designated carbon atom on an adjacent monosaccharide and is typically designed as an (L) as will be understood by a skilled person. The glycosidic linkage is denoted by α or β (m→n), wherein α or β represents a configuration of an anomeric carbon m of a monosaccharide and n represents the corresponding carbon on an adjacent monosaccharide to which the glycosidic linkage is connected. Polysaccharide in the sense of the disclosure range in structure from linear to highly branched and are often quite heterogeneous, containing slight modifications of the repeating unit. Polysaccharides, have a general formula of C_(x)(H₂O)_(y) where x can be a number between 200 or lower and 2500 or higher.

In embodiments, herein described the antigenic polysaccharide component from a Francisella bacterium capable of triggering a humoral immune response comprises one or more antigenic polysaccharides from the Francisella bacterium and/or one or more derivative thereof. The one or more antigenic polysaccharides from the Francisella bacterium and/or one or more derivative thereof can be isolated from the Francisella bacterium or produced synthetically to include the type and number of saccharides of the polysaccharide from the Francisella bacterium or derivative thereof. Antigenic polysaccharides can be chemically synthesized through chemical polymerization using linear or convergent approaches as will be understood by a person skilled in the art.

In some embodiments, a Francisella polysaccharide component can comprise an antigenic Francisella polysaccharide having a Formula (XI):

wherein

M1-M4 can each independently be selected from the group consisting of (Xa) to (Xw):

L1-L4 can be selected independently from the group consisting of:

α (1→2), α (1→3), α (1→4), α (1→5), α (1→6), β (1→2), β (1→3), β (1→4), β (1→5), and β (1→6); and

n is 1 to 1250,

-   or a derivative thereof.

Derivatives of an antigenic Francisella polysaccharide of formula (XI) typically comprise at least one or two epitopes of the antigenic Francisella polysaccharide.

Epitopes of an antigenic polysaccharides such as the O-polysaccharide from Francisella can be provided by two tetrasaccharide repeats for antibodies recognizing internal O-Ag epitopes, and a single repeat for antibodies recognizing the terminal O-Ag [10] wherein a structural characterization (and molecular docking simulation) of the interaction between an internal O-Ag binding antibody and a 2-unit tetrasaccharide molecule. If the tetrasaccharide unit is represented as “a, c, b, d” (where each letter is a different monosaccharide comprising the tetrasaccharide, and “a, b, c, d, a, b, c, d” represents a 2-unit tetrasaccharide, the binding epitope for this particular Ab would be 6 residues represented as “b, c, d, a, b, c” [11].

In some embodiments, the antigenic polysaccharide of formula (XI) is a polysaccharide isolated from a Francisella bacterium as a polysaccharide molecule (e.g. as a Capsular Polysaccharide). In some embodiments, the antigenic polysaccharide of formula (XI) is a polysaccharide isolated from Francisella bacterium within a multicomponent Francisella molecule (such as lipopolysaccharide) which further comprises additional molecular components of a different chemical nature such as lipid or protein components.

A derivative of the antigenic polysaccharide of formula (XI) can be obtained from the polysaccharide following isolation of the polysaccharide from a Francisella bacterium whether said polysaccharide is isolated as a polysaccharide molecule or as a part of a multicomponent molecule. In particular, in some embodiments, a derivative of the antigenic polysaccharide of formula (XI) can be any moiety obtained from fragmentation (e.g. by hydrolysis or other suitable processing) of the polysaccharide molecule, and/or multicomponent molecule from the Francisella bacterium. In those embodiments the derivative also has an ability to trigger a humoral immune response which can be the same lower or higher than the one of the base antigenic polysaccharide of formula (XI).

In some embodiments, a derivative of the antigenic polysaccharide of formula (XI) can be obtained by synthetically producing any moiety of the polysaccharide of formula (XI). In particular, in some of these embodiments, a derivative of the antigenic polysaccharide of formula (XI) comprises at least one tetrasaccharide repeat unit comprising monomers selected from the group consisting of Xa to Xw.

Polysaccharides and derivatives thereof can be chemically synthetized through chemical polymerization using linear or convergent approaches as will be understood by a person skilled in the art. For example, the polysaccharides can be extended by one monosaccharide unit selected from the group consisting of Xa to Xw at a time. An oligosaccharide can be build starting from either the non-reducing or the reducing end. The resulting disaccharide can be converted either into a new glycosyl donor or into a new glycosyl acceptor. The disaccharide is then coupled with another monosaccharide building block to provide a trisaccharide. The process is reiterated to provide a single tetrasaccharide of Formula (XI) or until an oligosaccharide of the desired length is obtained. Alternatively, in the convergent approach, tetrasaccharide building blocks are synthesized separately and subsequently used for the assembly of a larger polysaccharide.

In some embodiments, the antigenic Francisella polysaccharide of formula (XI) or of derivative thereof, can comprise one, two or more repeats of the tetrasaccharide of Formula (XI).

In some embodiments, herein described in the antigenic Francisella polysaccharide of formula (XI) or of derivative thereof, n can be 1 and the antigenic Francisella polysaccharide can be a tetrasaccharide as will be understood by a skilled person. The term “tetrasaccharide” as used herein refers to a moiety having four monosaccharide units joined sequentially by a glycosidic linkage.

In some embodiments, in the antigenic Francisella polysaccharide of formula (XI) or derivative thereof, M1-M4 can each independently be selected from the group consisting of α-D-Glc (Xc), α-D-GalNAc (Xm), α-D-GlcN (Xe), α-D-GlcNAc (Xg), α-D-GalNAcAN (Xq), β-D-QuiNAc (Xs), β-D-Qui2NAc4NAc (Xu), and β-D-Qui4NFm (Xw). In some preferred embodiments, M1-M4 are the following saccharides in this specific order: β-D-Qui4NFm (Xw), α-D-GalNAcAN (Xq), α-D-GalNAcAN (Xq), and β-D-QuiNAc (Xs).

In some embodiments, in the antigenic Francisella polysaccharide of formula (XI) or derivative thereof, M1-M4 can each independently be selected from the group consisting of α-D-GalNAc (Xm), β-D-QuiNAc (Xs), and β-D-Qui4NFm (Xw). Preferably M1 and M2 are the same, and more preferably M1 and M2 are α-D-GalNAc (Xm).

In some embodiments, in the antigenic Francisella polysaccharide the antigenic polysaccharide of formula (XI) or derivative thereof, L1, L2, L3 and L4 each independently be selected from the group consisting of α (1→4), α (1→3), β (1→2), and β (1→4), respectively. Preferably, L1 and L2 can be independently a (1→4), and a (1→3) and L3 and L4 can be independently β (1→2), and β (1→4).

In some embodiments, in the antigenic Francisella polysaccharide the antigenic polysaccharide of formula (XI) or derivative thereof, L1, L2, L3 and L4 each independently be α (1→4), α (1→3), β (1→2), and β (1→4), respectively.

In some embodiments herein described the antigenic Francisella polysaccharide the antigenic polysaccharide of formula (XI) or derivative thereof, n can be 1 to 10, 10 to 50, 50 to 100, 100 to 250, 250 to 500, or 500 to 1,000.

In some embodiments of the antigenic combination herein described, the antigenic Francisella polysaccharide or derivative of Formula (XI) can be an antigenic fragment of Francisella LPS herein described where the antigenic Francisella polysaccharide or derivative of Formula (XI) may or may not include an additional lipidic component.

In some embodiments herein described, the antigenic Francisella polysaccharide of formula (XI) or derivative thereof is a polysaccharide molecule which further comprises a lipidic component. In some of these embodiments, the antigenic Francisella polysaccharide herein described is an antigenic lipopolysaccharide (LPS) from a Francisella bacterium capable of triggering a humoral immune response or a derivative thereof.

The term “lipopolysaccharide” or “LPS” as used herein refers to an integral component of gram-negative bacteria cell walls. In particular, LPS is a macromolecule of molecular mass about 10-200 kDa and is composed of a lipid component (also referred to as “Lipid A”) and a polysaccharide component comprising a core oligosaccharide attached to the lipid component by an eight-carbon sugar, 3-deoxy-D-manno-octulosonic acid (KDO) and an O-polysaccharide (also known as O-antigen), which contains a varying number of tetrasaccharide repeating units, typically from two to six sugars.

The lipid component of an LPS molecule or Lipid A is typically formed by a β-glucosamine-(1→6)-glucosamine-1-phosphate base with fatty acid esters attached to both carbohydrates. The acyl chain length and number of acyl groups can vary between bacterial species but are relatively conserved within a species. In an LPS molecule of a gram-negative bacteria the Lipid A typically anchors the LPS to the outer membrane. In Francisella bacteria lipid A typically has four long acyl chains with 16 and 18 carbons, whereas most bacterial lipid A has six acyl chains that are usually shorter, 12-14 carbons. Some Francisella lipid A exhibits temperature-regulated heterogeneity in their acyl chains (see Example 1 reporting exemplary LPS from Exemplary Francisella species).

The polysaccharide component of an LPS molecule typically includes an inner core and an outer core as well as an O-antigen portion. The inner core of the polysaccharide component typically contains between 1 and 4 molecules of the 3-deoxy-α-D-manno-octulosonic acid (KDO) attached to the disaccharide core. KDO is specifically associated with lipopolysaccharide, and biologically active lipid A is thought to require at least one KDO residue for bacterial survival. However, an Escherichia coli K-12 suppressor strain that is KDO deficient demonstrates that the KDO requirement is not absolute for viability. Typically, the inner core of the LPS polysaccharide component is also modified with heptulose (ketoheptose) monosaccharides, the most common of which is L-glycero-α-D-manno-heptopyranose. The inner core glycan residues are typically phosphorylated or modified with phosphate-containing groups, e.g., pyrophosphate or 2-aminoethylphosphate. The phosphate groups of lipopolysaccharides increase the overall negative charge of the cell membrane and help to stabilize the structure. Typically, the outer core of the polysaccharide component of the lipopolysaccharide contains more common hexoses, including glucose, galactose, and N-acetylglucosamine and is structurally more diverse than the inner core. In Francisella LPS the core is relatively small, and typically contains two mannose sugars in the inner core instead of the more commonly found heptoses. Francisella LPS differs from presence of different residues in this portion (see Example 1). Francisella LPS typically also has a single non-branched Kdo in its core region even though studies have shown that Francisella initially synthesizes its LPS with two Kdo sugars, one of which is removed by a novel Kdo hydrolase. The O-antigen portion comprises one or more repetitive units of an O-antigen which is a repeating oligosaccharide unit typically comprised of two to six sugars. The O-antigen is the primary structural constituent of lipopolysaccharide that differentiates bacteria. The distinctive O-antigen structures have been used to identify and assign serogroups to Escherichia coli, Salmonella enterica, and Vibrio cholerae. Lipopolysaccharides from rough mutant strains of E. coli lack the O-antigen portion of the structure. Studies aimed at gaining insight into anti-Ft LPS antibodies demonstrated that four monoclonal anti-Ft LPS antibodies all are specific for the O-antigen of Ft LPS [12].

The polysaccharide section and the lipid A section of a lipopolysaccharide can have variability in structure, while the O-antigen has a high degree of structural variability as well as variability in the number of repeating units. These differences result in a degree of heterogeneity in LPS preparations.

LPS molecules are variable in length (and molecular weight), due to variability in the number of repeating O-antigen oligosaccharide side chains. The molecular weights of individual F.t. LPS molecules range from ˜10,000 Da to >200,000 Da (as determined by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE).

Immune responses directed against Francisella LPS can be assessed by quantifying anti-Ft LPS antibodies present in serum from infected and/or vaccinated animals using standard Enzyme Linked Immunosorbant Assays (ELISA). Detectable antibody titers against Ft LPS would indicate that the molecule was immunogenic.

In some embodiments, the LPS from Francisella can be represented by a general formula (XZY)

X-Z-Y   (XZY)

wherein X is the O antigen, Z is the inner oligosaccharide or core forming with the O antigen the polysaccharide component and Y is the Lipid A component.

In particular, in some embodiments, the O antigen X has formula

in which M1-M4 can each independently be selected from the group consisting of (Xa) to (Xw):

in which L1-L4 can be selected independently from the group consisting of:

-   α (1→2), α (1→3), α (1→4), α (1→5), α (1→6), β (1→2), β (1→3), β     (1→4), β (1→5), and β (1→6); and -   n is 1 to 250.

In LPS of Formula XZY, the core Z is a chemical moiety linking a lipid A to an O antigen. In some embodiments, a Z can be a trisaccharide represented by formula

wherein

-   R30 to R32 are each independently a hydrogen H or represented by     Formula (Z11):

wherein M5 a monosaccharide selected from the group consisting of monosaccharide represented by Formulas (Xa) to (Xw), and R40 is a hydrogen H or a monosaccharide selected from the group consisting of monosaccharide represented by Formulas (Xa) to (Xw), wherein M5 and R40 are linked by linkage L5 selected from the group consisting of:

-   α (1→2), α (1→3), α (1→4), α (1→5), α (1→6), β (1→2), β (1→3), β     (1→4), β (1→5), and β (1→6).

In LPS from Francisella of formula XZY, the LPS further comprises a lipid A component Y. In some embodiments, Y is as a chemical moiety having at least one C12 to C18 linear or substituted acyl chain. In some embodiment, A lipid A can have two to six (2-6) C12 to C18 linear or substituted acyl chains.

In further embodiments, a lipid A Y can be the two to six (2-6) C12 to C18 linear or substituted acyl chains can be covalently connected to a disaccharide as represented by Formula (Y1):

wherein

-   R10 to R13 are each independently represented by hydrogen H or     Formula (Y11):

wherein R30 is hydrogen H, OH or represented by Formula (Y12):

R20 and R21 is represented by Formula (Y10):

wherein M10 is hydrogen H or a monosaccharide linked to the oxygen by the corresponding anomeric carbon, the monosaccharide is selected from the group consisting of Formulas (Xa) to (Xw).

Reference is made to the schematic illustration of FIG. 1 where the different components and their arrangement in LPS molecules are shown and to Example 1 and Example 2 describing exemplary LPS and related biosynthesis.

In some embodiments, the O antigen X of formula (XII) can have n ranging from 10 to 250, and preferably from 10 to 100.

In some embodiments, the O antigen X of formula (XII) can have M1-M4 can each independently be selected from the group consisting of α-D-Glc (Xc), α-D-GalNAc (Xm), α-D-GlcN (Xe), α-D-GlcNAc (Xg), α-D-GalNAcAN (Xq), β-D-QuiNAc (Xs), β-D-Qui2NAc4NAc (Xu), and β-D-Qui4NFm (Xw).

In some embodiments, the O antigen X of formula (XII) can have M1-M4 can each independently be selected from the group consisting of α-D-GalNAc (Xm), β -D-QuiNAc (Xs), and β-D-Qui4NFm (Xw). Preferably M1 and M2 are the same, and more preferably M1 and M2 are α-D-GalNAc (Xm).

In some embodiments, the O antigen X of formula (XII) can have L1, L2, L3 and L4 each independently be selected from the group consisting of α (1→4), α (1→3), β (1→2), and β (1→4), respectively. Preferably, L1 and L2 can be independently α (1→4), and α (1→3) and L3 and L4 can be independently β (1→2), and β (1→4).

In some embodiments, L1, L2, L3 and L4 of the O antigen X of formula (XII) can be α (1→4), α (1→3), β (1→2), and β (1→4), respectively.

In some embodiment, free lipid A as described herein can be represented by Formula (Y1a):

wherein R10, R11, R12 and R13 are defined as for Formula (Y10).

In some embodiments, the LPS comprises a tetrasaccharide repeat of (Qui4NFm-GalNAcAN-GalNAcAN-QuiNAc)_(n), with Qui4NFm at the non-reducing end and n between 1 and 100.

In some embodiments herein described an antigenic Francisella polysaccharide further comprising a lipid component, can comprise an antigenic fragment of Francisella LPS which comprises at least the O-antigen domain. In some embodiments, an antigenic Francisella LPS fragment can further comprise an LPS core polysaccharide herein described in addition to the O-antigen. In some embodiments, an antigenic Francisella LPS fragment can further comprise both a lipid A and a core polysaccharide herein described in addition to the O-antigen. An antigenic Francisella LPS fragment in accordance with the disclosure is capable of triggering a humoral immune response at a same, reduced or increased level compared to the Francisella LPS as will be understood by a skilled person.

In some embodiments, an antigenic Francisella LPS or fragments thereof herein described can be purified from any strain of Francisella tularensis. In some embodiments, Francisella LPS can be derived from Francisella tularensis subspecies holarctica Live Vaccine Strain (LVS). General methods of isolating LPS from a bacterial cell membrane can be found in [13-16], each incorporated by reference in its entirety.

One of the most common methods for isolation of LPS from bacterial cell walls involves disruption of the cell wall by sonication followed by proteinase and nuclease treatment. A hot phenol-mediated extraction step follows, which eliminates any residual amounts of enzymes added previously. Briefly, bacteria are disrupted by an overnight incubation with shaking at 37° C. in a buffer (pH 8.0) containing Lysozyme, followed by incubation with micrococcal nuclease. LPS is then precipitated and washed using Ethanol/Sodium Acetate. Purified LPS can be further characterized by SDS-PAGE electrophoresis followed by silver and commassie blue staining and HPLC according to techniques identifiable by a skilled person. Additional methods can be identified by a skilled person upon reading of the present disclosure.

In some embodiments herein described, the antigenic Francisella polysaccharide or derivative of formula (XI) can be an antigenic capsule polysaccharide (CPS) from a Francisella bacterium or a fragment thereof which is capable of triggering a humoral immune response or a derivative thereof.

The term “capsule polysaccharide (CPS)” as used herein indicates a macromolecule attached to the surface of Francisella having a molecular weight higher than 100 kDa and composed of a lipid component and a polysaccharide component comprising a varying number of tetrasaccharide repeating units. The characteristics of F. tularenesis CPS include 1) phenol resistance, 2) protease resistance, 3) ethanol precipitable, 4) high molecular weight (>100 kDa).

In some of those embodiments, the structure and composition of F. tularensis CPS can have a same or similar structure of the LPS O-antigen and therefore have formula

in which M1-M4 can each independently be selected from the group consisting of (Xa) to (Xw):

in which L1-L4 can be selected independently from the group consisting of:

-   α (1→2), α (1→3), α (1→4), α (1→5), α (1→6), β (1→2), β (1→3), β     (1→4), β (1→5), and β (1→6); and -   n is 1 to 1000.

In some embodiments, the CPS comprises a tetrasaccharide repeat of (Qui4NFm-GalNAcAN-GalNAcAN-QuiNAc)n, wherein n is greater than 100.

In particular, in some embodiments, F. tularensis capsule polysaccharide can comprise a tetrasaccharide repeat unit such as 2-acetamido-2,6-dideoxy-O-D-glucose (O-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (O-Qui4NFm), and/or 2-acetamido-2-deoxy-O-D-galacturonamide (O-GalNAcAN). In some embodiments, the F. tularensis CPS tetrasaccharide can have a molecular mass of approximately 790 Da, comprised of the following saccharides in this specific order: β-D-Qui4NFm (Xw), α-D-GalNAcAN (Xq), α-D-GalNAcAN (Xq), and β-D-QuiNAc (Xs).

In some embodiments of F. tularensis CPS, the tetrasaccharide units of formula (XII) are further covalently linked to a lipid component. The CPS lipid moiety has the structural properties characteristic of F. tularensis lipid A, but with a composition distinct from the free lipid A. In particular, the CPS Lipid A-like anchors have molar ratios of 3-OH-18:0/3-OH-16:0 less than 3 compared to >5 for Lipid A or LPS. The most prominent non-hydroxylated fatty acid present in CPS LipidA-like anchors is C14:0 compared to C16:0 for LipidA or LPS.

Methods of extraction and purification of a F. tularensis CPS are identifiable by a skilled person as well as methods for the related characterization. A specific example can be found in related references such as in Apicella et al. [17] however other procedures can be used as will be understood by a skilled person (see also Example 19 of the present disclosure).

In some embodiments, the antigenic Francisella polysaccharide component can comprise more than one antigenic polysaccharide from the Francisella bacterium or derivative thereof in the sense of the disclosure. In particular in some embodiment the antigenic Francisella polysaccharide to be included in the antigenic combination of the disclosure can comprise an antigenic Francisella lipopolysaccharide (LPS) and/or a fragment thereof and an antigenic capsule Francisella polysaccharide (CPS) or a fragment thereof in various combinations as will be understood by a skilled person.

In antigenic combination herein described an antigenic Francisella polysaccharide component is comprised together with at least one protein antigen component from the Francisella bacterium.

The term “protein antigen” refers to a protein that is an antigen with respect to the immune system of an individual. In embodiments herein described, a protein antigen incorporated in the antigenic composition is a protein antigen isolated from a Francisella bacterium or recombinantly produced to obtain protein antigen with the primary, secondary and tertiary structure of a protein antigen isolated from a Francisella bacterium as will be understood by a skilled person.

In embodiments herein described a Francisella protein antigen component comprises one or more Francisella protein antigens and/or one or more derivatives thereof.

Suitable Francisella protein antigens in the sense of the present disclosure comprise any protein antigen from a Francisella bacterium that can confer cell mediated immune response. In particular, in some embodiments, suitable Francisella protein antigens comprise antigens capable of inducing a robust T cell response in vivo in an INF-γ dependent manner. A variety of in vitro and in vivo assays such as cell-based assays and assay in animals, can be used for identification and selection of protein antigens as will be understood by a person of ordinary skill in the art upon reading of the present disclosure.

In some embodiments, the Francisella protein antigen comprised in the antigenic combination can be selected from a group consisting of IglC, DnaK, OmpA, SucB, LpnA, FopA, Lpp3, Type IV pilus fiber building block protein, FopB, FTT_1676, FTT_1441, and FTT_1778c.

A list of exemplary coding sequences of the above-mentioned Francisella protein antigens from Francisella tularensis subsp. tularensis SCHU S4 is reported in Table 1 below. The sequences are available from public databases such as NCBI gene database (see e.g. the web addresses for the exemplary sequences indicated in the table) at the date of filing of the present disclosure as will be understood by a person of ordinary skill in the art.

TABLE 1 Exemplary Francisella protein antigen gene sequences from Francisella tularensis subsp. tularensis SCHU S4 SEQ ID Number of Annotation Gene sequences NO TM helices Ig1C1 and atgattatgagtgagatgataacaagacaacaggtaacaagtggcgagaccattc  1 0 Ig1C2 atgtgagaactgatcctactgcatgtataggatctcatcctaattgtagattatt (FTT1357c and tattgattctttaactatagctggggagaaacttgataaaaatatcgttgctata FTT_1712c):  gatggtggagaggatgtcacgaaagctgattcggctacagctgctgctagtgtaa https://www. tacgtttatctataacgccaggctctataaatccaacaataagtattactcttgg ncbi.nlm.nih. tgttctaattaaatcaaatgttagaactaaaattgaagagaaagtttcgagtata gov/gene/?term= ttacaagcaagtgctacagatatgaaaattaagttaggtaattctaataaaaaac ig1C+AND+ aagagtataaaactgatgaagcatggggtattatgatagatctatctaatttaga francisella gttatatccaataagtgctaaggcttttagtattagtatagagccaacagaactt atgggtgtttcaaaagatggaatgagatatcatattatatctatagatggtctta caacatctcaaggaagtttgccagtatgttgcgcagctagcacagataaaggagt tgctaaaataggatatattgcagctgcatag DnaK ATGGGAAAAATAATAGGTATAGATTTAGGTACTACTAACTCTTGTCTTGCTATTA  3 0 (FTT_1269c) TGGATGGCAAGACTGCTAAAGTTATTGAGAATGCTGAAGGACATAGAACAACACC https://www. TTCAGTTGTGGCATATACTGATAGCGGTGAAATATTAGTAGGTCAAGCTGCTAAA ncbi.nlm.nih. AGACAAGCTGTAACTAACCCTGATAATACATTCTTTGCTATCAAGAGACTTATAG gov/gene/3191531 GTCGTAAGTACGATGATAAAGCTGTACAAGAAGATATTAAAAAGAAAGTACCTTA TGCGGTAATTAAAGCTGATAATGGTGATGCTTGGGTTGCTACTAAAGAAGGCAAA AAAATGGCTCCACCACAAGTTTCTGCAGAAGTTCTAAGAAAAATGAAAAAAACAG CAGAAGACTATCTAGGTGAACCAGTTACAGAAGCTGTAATTACAGTGCCAGCATA CTTTAACGATAGTCAAAGACAAGCTACAAAAGATGCTGGTAAAATAGCAGGTCTT GAAGTTAAAAGAATTATCAACGAGCCTACAGCGGCAGCGCTGGCATATGGTGTAG ACTCTAAGAAAGGTGAGCAAACTGTAGCGGTGTATGACCTAGGTGGTGGTACATT CGATATCTCAATTATTGAGATTGCTGATGTTGATGGCGATAACCAAATCGAAGTA TTATCAACCAATGGTGATACTTTCTTAGGTGGTGAAGACTTCGACTTGGCTTTAA TGAACTATCTAATTGACGAGTTCAAAAAAGAGCAAGGTATAGATCTTCACAATGA TAAGCTTGCTTTACAAAGAGTTAGAGAGGCTGCTGAGAAAGCTAAAGTAGAATTA TCTTCAGCACAACAAACTGATGTTAACCTACCTTACATCACAGCAGATGCTACTG GACCTAAGCACTTAAATATCAAAGTAACTAGAGCTAAGTTTGAGTCTTTAGTTTC TGATCTTGTAATGAGATCACTTGAGCCTTGTAAGAAAGCTCTTGAAGATGCTGGT TTAAGTAAGTCTGATATTACAGAAGTATTACTAGTGGGTGGACAAACTCGTATGC CTCTAGTACAAGAGAAAGTAAAAGAGTTTTTTGGTAAAGAGCCACGTAAAGATGT GAACCCTGATGAAGCTGTTGCAGTTGGTGCGGCTATTCAAGGTGGTGTATTAGCA GGTGATGTTAAAGATATTCTTTTATTGGATGTAACACCGCTTTCTCTAGGTATTG AGACTATGGGAGGTGTTATGACTAAGCTTATCGAGAGAAATACTACGATTCCTAC TAAGAAGTCGCAAGTATTCTCAACAGCTGAAGATAACCAGCCTGCGGTAACTATT CATGTACTTCAAGGTGAGCGTGAAATGGCTTCTGCAAACAAATCTTTAGGTAGAT TTGATCTGGCAGATATTCCACCAGCGCCACGTGGTATGCCACAAATTGAGGTTAC TTTTGATATAGATGCTAACGGTATATTAAATGTGTCTGCTAAAGATAAAGCTACT GGTAAAGAGCAAAATATTGTGATTAAGTCTTCAAGTGGTTTATCTGAAGAGGATA TCGAAAAAATGGTACAAGACGCTGAAGCTAATGCAGAAGCAGATAAAAAGTTCCA TGATTTAGTTACTGCTAGAAATACTGCTGATAACTTAATTCATAGCTCAAGAAAA GCAATTCAAGAACTGGGTGACAAAGTAACAGCAGCAGAAAAAGAAAAAATCGAAG AAGCTTGTAAAGAGCTTGAAGCAGCAACTAAAGGTGATGATAAGCAAGCGATTGA ATCTAAAACTAAGGCTCTAGAAGAAGCATTTGCGCCAATAGCTCAAAAAGCTTAT GCTGAGCAAGCTCAAGCTGCTGTTGCCCAAGGTGGTGCTAAAGCTGAAGAACCTA AGAAAGAAGAAGATGTTGTTGATGCTGACTTTGAGGATGTTGAAGACGACAAAAA ATAA OmpA ATGAAAAAATTACTGAAACTATGCTTAATGACATCATTAATTACAACACTCTCAG  5 0 (FTT_0831c) CCTGCCAAACACTAGATGATAAAGATAAAGATAGTGGTCCACTAACATTTCCAAC https://www. ATTAGAACCTTGTACTGCCGAGCTACTTCAATCAAATCAATCTTTTATATGTGTA ncbi.nlm.nih. AAAGAACAAACAGGACCTGATCTAATCGAAACAAATATAAAATTTGATGCTGATA gov/gene/3191752 GCTATACATTAAACACTCAGGCTAAAGAAGTTTTAGATAAGCTTTTTGCTTATTT GAAACTAACTGATACTACAAATTTCACAATTAAAGGTTATGCTGGAAAAGTTGAA TCAAAAATTCTCACAGATCAGAAAATCCTAACCGACTATAATATTAGACTATCAA AAAACCGTGCTAGTAGTGTCGAAGAATACCTTGTAAACAAAGGTCTTGGCTCTAG TGATGGAATTACTATTAAAGCCTTAGGTTATCAAGATCCTATCGCCCCTAATGAC TCAACCTCAAGTAGAGCTATAAATCAGCGCGTTGAAATTACTCTAAAAAGTAGAC TTATAGAGCAAATTGATAATATTGAAAATAACTTAGAGCATGTCAGACCAGCTGA ATATACAAAATTCTTCTCAAATGTATATTTACTTAATGATAATCAGATAGACAAT ATTTCAAGAATATACAATTCTAGAGAGAAACGCCCAATACTCGGAATTAACTTTA AAATCTTTGCTAACAAAGAATATACAGCAGCCAAGGATAACAGTAACTTCATAAT AATATCCGAACCAAAACCAATATCTTCATTTAATGATGATAAAAAAGTCTATAGA CTAGGCTCAGCAAAATATGATTATACCTTTAAAGGTATAACAGCATTGACGATAA CTAATTTAAGTCGTGAAGCTAGTGTAGGCAATTATGTAATACCTAATGATATTGT TTCACAACAACTGCCAGAACAAACTTTTAAAATGAAAAGTAAAATAACAGCTAAT GTACTTGAAGATGTAATGAATACTAATACATTCTCATCTTCGTATAATAGTATTC TATTGAACAAAGGCGCTGCTGATGGCTTGAAAGTAGGCGCTCAAGTTATTTTATA TGAACCAGAAACCAGAGTAGATGGTTTTCCAGTCCCACCTAAATATATTGGTTAT GGTTTTATCTATAGAGAATCTCAACACTACTCTATAGCCCTAATTGTCAATTCAC TACAAGAAATTACAAATAATTCAATGGCAACGACTATTTTATAA SucB ATGGTTGAATTAAAAGTACCTATGTTCCCAGAGTCTGTAGCAGATGGCACATTAG  7 0 (FTT_0077) CTCAATGGAATAAAAACGAAGGTGACTTTGTAAATGAGGGCGATATCTTGGCAGA https://www. GATTGAGACTGATAAAGTTGTTCTAGAAGTACCTGCAACATCTAGTGGTGTTTTA ncbi.nlm.nih. AAAGGGATAAAAAAACATGCTGGTGATACAGTGCTTTCAGAAGAGTCATTAGCGA gov/gene/?term= TCATTGATACTGCTGTTTCTACATCTGAACCTAACCAACAAACTACTAATCAAGG SucB+ AAATGCTTCAGAAGCAACTGCTACTGGGCAAGAAATTGATATTAAGGCGCCTGTA francisella TTTCCAGAGTCTGTAGCAGATGGCACGATCTCAGAGTGGCATAAGAAAGAGGGTG AGGCTGTTTCTGAGGGTGATATCTTAGCAGAGATTGAGACTGATAAGGTTGTTCT AGAGGTTCCGGCAACATCAAATGGTGTTTTGACAAAAATATTAAAAACAGCAGGA GAGACTGTACTATCTGCAGAGCTTATCGCTAAGATTACAGCAGGAGGCGCAACTG CTACTACGAAATCAGAAGCTTCGGTGGGAGTTTCTCAAGCAAATAATGATCCGCA TCTAGTACCTTCAGCACGTAAAGCTTTTAATGCAAGCGGCTTGGATACTGCTGCT AATATCGAAGGTACAGGTAAAAAAGGGCGTATAACTTCTGAAGATGTCAAAAAAG CAGTTGCATCAGTAAATAAACCTCAACAACAGACAGTTGTTATAAATCAAGGTGC TAGATATGAAAAAAGAGTCAAGATGACTCGTCTGCGTCAGACTATAGCAAATAGG TTAGTTGAGGTTCAACATACTAATGCAATCTTAACTACTTTCAATGAAGTAGATA TGAGTGCAGTTATGGAGCTTAGAAACAAATATAAAGATATGTTTGTCAAAGAACA TGATACTAAGCTTGGCTTTATGTCTTTCTTTATCAAAGCAGCAACAGAAGCACTT AAGAAATTCCCAGATGTAAATGCCTCTATTGATGGTGATGAGATTGTTTACCATA ATTATTTTGATATTGGTATTGCTGTAGGTACTGATAGGGGTCTAGTGGTACCTGT ACTAAGAGATACAGATACTAAATCTCTAGCTGAATTAGAAGCCGATGTTTTAGAC AAAGCGATTAAAGGTCGTGATGGTAAATTAAGCCTTGAAGATATGCAAGGTGGTA CATTTACGATTACAAATGGCGGAACTTATGGTTCGATGTTATCTACGCCTATTAT TAATTCACCGCAAAGTGCTATTTTAGGTATGCATAATATTGTTGAGCGTCCTGTA GTTGTTAAGGGTGAGATTAAGATTCGTCCAATTATGTATTTAGCGTTATCTTACG ACCATAGAATCATTGATGGCGGTACATCTGTAAGATTCTTGAAGATGATCAAAGA GCTAATTGAAGATCCAAATAGAATTCTTCTACAAGTATAG LpnA/Tu14 ATGAAAAAAATAATTGAGCTTAGTCTTTTATCTTTATCAATCGCAGGTTTAGCGA  9 1 (FTT_0901) GCTGTTCTACTCTAGGGTTAGGTGGCTCTGATGATGCAAAAGCTTCAGCTAAAGA https://www. TACTGCTGCTGCTCAGACAGCTACTACTGAGCAAGCTGCTGCTGTATCTAAGCCA ncbi.nlm.nih. ACTGCAAAAGTAAGTTTAAATAAACTTGGTCAGGATAAAATAAAAGCAACTGTAT gov/gene/3191792 ATACAACATACAATAATAACCCACAAGGAAGTGTAAGATTACAATGGCAGGCTCC AGAAGGTTCTAAGTGCCATGATACAAGCTTCCCAATTACTAAGTATGCTGAGAAG AACGATAAAACTTGGGCAACTGTAACAGTTAAGCAAGGTAATAACTTCTGTAGCG GTAAGTGGACAGCTAATGTAGTTTATGACAAAGAAGTAATCGCTTCTGATTCAAT AAATATTTAA FopA1 TTGATGAGATTAAAAAGTATTGTTATAGCTACAACTGTATTATTAGGTTCAGCTA 11 1 (FTT_0583) CAGCATCTATCGCTGCAGGTTCAGATAATATCGATACATTAGCAAACACTAATTC https://www. AGCTACTACACAAAGCAGTGGTTTTGCAGCTAATAATTTCATTGCTCCTTTTGCA ncbi.nlm.nih. AATACTTATAGCGCTTTGACTAACAAGGACAATACTTGGGGTCCTCAAGATAGAA gov/gene/3192005 CTGGCCAGTGGTACTTAGGTGTAGATGCTAACGGTCTAGCTGGAACTCCTAACTC TCCATCAGGTGCTGGTGCTAACTTCACAATCGGTTATAACATCAATAAATACTTC GCTGTACAGTACAACCAATTAGTTGGTAGAGTATTTGCTGGTTTAGGTGAAGGTG TTGTAAACTTTAGTAATAATACTATGTTTACTCCATATGCTGCAGGTGGTGCTGG TTGGGCAAATCTAGCAGGTCAAGCAACAGGTGCTTGGGATGTGGGTGGTGGTCTT AAGTTTGAACTATCTAGAAATGTTCAAGCAAGTGTTGACTACAGATATATCCAAA CAATGGCACCTAGTAATATTTCTGGTGCTAATGGCAGAGCGGGTACTAACATGAT TGGTGCTGGTTTAACATGGTTCTTTGGTGGCAAAGATACTACTAATAATGACACT GGTAATATTCAGGATAATGGTGCGACTACAGCTGCTCAAACTGTTGCTATGCCAA CTATTGATGAGTCTAAGTATGTTTTACCTGCTGGTATTAAGCAATGTGAAGGCAA CTTTAATCTAACTGAAGATGGTGTCGCGTGCTATACAATAAATGGTGATGATGTA ACAGTTTACCTAGATACTAAGTTTGCTTATGATAAAGCTACTTTAAATGCTAAAG GTAAAAAAGCTATTGCATCTTTTGTTAATTTTATCAAGGATAGTAACATTAGCTC TGTAACAGTTAAAGGTTATGCTTCTCAAGGTCAAACTGGTAGCGAGTTTGATATA TATAACCAAAAACTTTCTGAGAAGAGAGCACAAGCTGTTGCTGATTACATGAAGC AATTAGGTTTAGATAGTGAGAAAATAATTACTAAAGGTTTTGGCTATAATGATAC TTTAGGTGGTATTCATAAGTCTGATCCGCGTAACCAGCGTGTAGAAGCTAGCGTA TCAGCTCCACTTAAAGAAGCTAACTAA Lpp3 ATGAAAGGATTAAAAGCAAAAATATATATAATTTTTTTGGCTGCAGTGCTAGCGG 13 1 (FTT_1416c) TTATTTCAGGCTGTGCTACTGACAAAGGAACCCAGTATAAAGATGGTTATTATAT https://www. AACTACGCTTAACTATAATTTTAATACGGTTTATAATGCTACTCTGCAAGCAATA ncbi.nlm.nih. CAAAACGGACAAACATTTGATTATAAGAGTAACCCATATGATATATCAGTTAACA gov/gene/3191965 AAAATAATGGAACTGATGCTGAGATAGTTTCTGCTAGTGATAGCGATTCTACAGA CTCTCTACAAGTAGCAATGAAGAAGTTACCTAATAATGCTACAAGGATTTCAATT AAATATGGTAGCCAAGGAAATTCGATTAGATCTTCAGCGCTAATAGGCATAATAG AAGGAAATATTCGCTACGCTAATACATAA Type IV pilus ATGAAAAAGAAAATGCAAAAAGGTTTCTCACTAGTTGAGTTAATGGTAGTGATCG 15 1 fiber CGATCATCGCTATCCTAGCAGCTGTAGCGATCCCGATGTACTCTAACTACACTAC building ACGTGCTCAGTTAGGCTCTGATCTATCTGCTCTAGGTGGTGCTAAAGCTACAGTA block protein GCTGAAAGAATAGCTAACAACAATGGTGATGCATCTCAAGTTACAATTCTTCAAG (FTT_0890c) CTAATGCCGCTGCAAATGGTCTTCCAAGTGGTGCTTCAGTTGCTGCTGGTACTAT https://www. TAGTTATCCATCAACAGTATCTGGTGCAACAATTCAATTAGCTCCTACAGTAAGT ncbi.nlm.nih. TCCGGTGCTATTACTTGGACTTGTAATATTTCAGGTGTATCAGCATCTCAAGTAC gov/gene/?term= CATCTAACTGTAATGCTATCTAA FTT_0890c+ francisella FopB ATGTTTTATAAACTAAACAAGCAAGGAAGAAAAATTATGAAAAAAACTATACTAG 17 1 (FTT_1747) GAGCAATGATTGCTGGTGGTTTGATGGTTTCTGCAACTACAGCTATGGCTGGTGG https://www. GGTAGGTTTTGCTAATGTCCAAGATATTTTTGAAACTTCACCTCTTGGTAAAGCA ncbi.nlm.nih. AAGGTAACAGCTGATGAGAAAAAGCTAAAGCCACAGATGGATCAACTTAAGCAAA gov/gene/?term= ACATCACTGCTTTACAAGAGAAAGTAAATGCATACACTCAAGAAAAAGATGATGT FTT_1747+ TCAAACAGATGATTCTAAAGGCGATACTAAAGATGCACAGTCAGCAGACAAAGTA francisella GAAAATCAAGATAAGCAACAAGCTCAAGCAGATCTTGAAAAAGCAATGAAAGATT ACCAAAACCTAATGAATCAGGTTCAAAAAATGGCTTCTGACGATGCTGATGCGTT CAAAGATGCTTTGACTAAGGCTTCTGCTCAAGTTGCTAAAGAGAAGCAACTTGAT GCTATTTTACCTGCTGAAATGAGTCTATATAATGTTGATAGTATTGATGTTACTA AAGATGTTATAGCTAAGATGCAATAA Hypothetical ATGGAATTTAGTATGCGCAATATATTCAAAAAAACATTATTAATATTATTACTAA 19 1 protein GCGTAATTATAGCATCATGTGGTATGCTATCTAACGACCAGTTTGTTTATCTAGG (FTT_1676) TCATGGTGAGAACTCTCCTGACTATCAGCTCTATTACGATAAGACACAAAAACTT https://www. TTTGTGCTAATTGATAAGAGAAATGGTTGTTTTCAGAAAGATGATACAGGTACTT ncbi.nlm.nih. GTCTTGCATTTACATTAAAACAAGCTCGTGAATTTAGAGAATATGTTTTAGCTAA gov/gene/?term= GATGATTGAAATAGATGTTAGACTTGCTAAGGACGACTATGGAAACTACGCTATT FTT_1676+ GAAGAGTTACGAAAAGCTGGGGTTACCACGGTTAATAAACCAATTAAAACTAAAA francisella AAGTTTTTGCAACTCCAGTTAAGCAGATTGTCCTTGATAGAAAGCAACAATATCA CCTTGTTAGAAAAGAATATGAAATAGATTCAAATCTTGTAGCAATGGTTGTTAAT GATAAAGAAGGTAAAAAACGTATAAAAGTTGCTTATACTGTTGATTTTCCTGGCT TAGAGAAAGAGTATAGTACCGAGCTTAGACCATTTATTATTGATCCAGAATACTT ATATACACATATGACTATTGACGCTGTCCATGAAGCTCAATTCATGCAGAGAGAT GTTATGAAGAGTCAAACAAATGTCAAAAAGAAAGTTGATGACTATCTTAAAGATG TTGTCGACAGCGATAAAAAAGATACAGGATACACTCACCAAGAAGTTGCAAGTAG TGTCGCAGCGCTAGATAACGATCTAATAACTAAGGCAGATGAGCAACGCGAAGAC AAAGAAACCACACTAGCAAGTGGTAGTAGTATTAGTACTATAGCGAAAAAGCCTA TCAATCAAGAAAGTAGTGGTAGTACAATACAAACAGCCACAAAAACTGCTTCAAC CACTCAAGTTGATAGCAATGATACTCCAAAAACAACTCTAGCAAGTGACAATAAA TAG Hypothetical ATGTTGATTATAATGATTAGAGTTTTAAATAATGGAGATAACAATATGGAACTTC 21 0 protein AATTAGAAAATAAACAAGAAATTATTGATCAATTAAATAAAATCTTAGAACTCGA (FTT_1441) AATGTCTGGAGTTGTGCGTTATACTCATTATTCTTTAATGATTATAGGTCATAAT https://www. AGAATTCCTATAGTTAGTTGGATGCAATCTCAAGCAAGTGAAAGTTTAACTCATG ncbi.nlm.nih. CTACTGCAGCAGGTGAAATGATAACTCACTTTGGTGAGCATCCATCTTTAAAAAT gov/gene/?term= AGCAGATTTAAACGAAACTTATCAGCATAATATCAATGATATATTAATCGAAAGT FTT_1441+ CTAGAACATGAGAAAAAAGCTGTTTCAGCATACTATGAACTTCTAAAACTTGTAA francisella ATGGCAAATCAATAATATTAGAAGAATATGCAAGAAAACTCATAGTTGAAGAAGA AACGCACATTGGTGAAGTAGAAAAAATGTTAAGAAAACCTGCATAA Hypothetical ATGAAATTAAGAAAAGTATTAATCGCGACATTATTAGGAGCTTCTGCTTTATCTT 23 1 protein TAAGTAGTTGTTGGTTACTTGTTGGTGCAGCTGTTGGTGGTGGAACTGCTGCGTA (FTT_1778c) TATTTCTGGTGAGTATTCAATGAATATGAGTGGCAGTGTAAAAGATATTTACAAT https://www. GCTACTTTAAAAGCTGTTCAAAGCAATGATGATTTTGTAATTACTAAAAAATCTA ncbi.nlm.nih. TTACTTCTGTTGATGCAGTTGTTGATGGTAGTATTAAGGTAGACTCAACAAGTTT gov/gene/?term= CTATGTTAAAATAGAAAAACTTACTGATAATGCTTCAAAAGTTACAATTAAGTTT FTT_1778c+ GGTACTTTTGGTGACCAAGCAATGTCAGCAACATTAATGGATCAAATCCAAAAGA francisella ATCTTTAA

A list of exemplary protein sequences of the Francisella protein antigens is reported in

Table 2 below. The sequences are available from public databases ((see e.g. the web addresses for the exemplary sequences indicated in the table) at the date of filing of the present disclosure as will be understood by a person of ordinary skill in the art.

TABLE 2 Exemplary Francisella protein antigen protein sequences from Francisella tularensis subsp. tularensis SCHU S4 SEQ ID Annotation Gene sequences NO FTT_1357c and MIMSEMITRQQVISGETIHVRTDPTACIGSHPNCRLFIDSLTIAGEKLDKNIVAIDGGEDVIKADS  2 FTT_1712c ATAAASVIRLSITPGSINPTISITLGVLIKSNVRTKIEEKVSSILQASATDMKIKLGNSNKKQEYK (Ig1C) TDEAWGIMIDLSNLELYPISAKAFSISIEPTELMGVSKDGMRYHIISIDGLITSQGSLPVCCAAST (https://www. DKGVAKIGYIAAA uniprot.org/ uniprot/Q5NEC5) FTT_1269c MGKIIGIDLGTINSCLAIMDGKTAKVIENAEGHRTIPSVVAYTDSGEILVGQAAKRQAVINPDNIF  4 (DnaK) FAIKRLIGRKYDDKAVQEDIKKKVPYAVIKADNGDAWVATKEGKKMAPPQVSAEVLAKMKKTAEDY (https://www. LGEPVTEAVITVPAYFNDSQRQATKDAGKIAGLEVKRIINEPTAAALAYGVDSKKGEQTVAVYDLG uniprot.org/ GGIFDISIIEIADVDGDNQIEVLSINGDIFLGGEDFDLALMNYLIDEFKKEQGIDLHNDKLALQRV uniprot/Q5NFG7) REAAEKAKVELSSAQQTDVNLPYITADATGPKHLNIKVIRAKFESLVSDLVMRSLEPCKKALEDAG LSKSDITEVLLVGGQTRMPLVQEKVKEFFGKEPRKDVNPDEAVAVGAAIQGGVLAGDVKDILLLDV TPLSLGIETMGGVMTKLIERNITIPIKKSQVFSTAEDNQPAVTIHVLQGEREMASANKSLGRFDLA DIPPAPRGMPQIEVTFDIDANGILNVSAKDKATGKEQNIVIKSSSGLSEEDIEKMVQDAEANAEAD KKFHDLVTARNTADNLIHSSRKAIQELGDKVTAAEKEKIEEACKELEAATKGDDKQAIESKTKALE EAFAPIAQKAYAEQAQAAVAQGGAKAEEPKKEEDVVDADFEDVEDDKK FTT_0831c MKKLLKLCLMTSLITTLSACQTLDDKDKDSGPLTFPTLEPCTAELLQSNQSFICVKEQTGPDLIET  6 (OmpA) NIKFDADSYTLNTQAKEVLDKLFAYLKLTDTTNFTIKGYAGKVESKILTDQKILTDYNIRLSKNRA (https://www. SSVEEYLVNKGLGSSDGITIKALGYQDPIAPNDSTSSRAINQRVEITLKSRLIEQIDNIENNLEHV uniprot.org/ RPAEYTKFFSNVYLLNDNQIDNISRIYNSREKRPILGINFKIFANKEYTAAKDNSNFIIISEPKPI uniprot/Q5NGK6) SSFNDDKKVYALGSAKYDYTFKGITALTITNLSREASVGNYVIPNDIVSQQLPEQTFKMKSKITAN VLEDVMNTNTFSSSYNSILLNKGAADGLKVGAQVILYEPETRVDGFPVPPKYIGYGFIYRESQHYS IALIVNSLQEITNNSMATTIL FTT_0077 MVELKVPMFPESVADGTLAQWNKNEGDFVNEGDILAEIETDKVVLEVPATSSGVLKGIKKHAGDTV  8 (SucB) LSEESLAIIDTAVSTSEPNQQTTNQGNASEATATGQEIDIKAPVFPESVADGTISEWHKKEGEAVS (https://www. EGDILAEIETDKVVLEVPATSNGVLTKILKTAGETVLSAELIAKITAGGATATTKSEASVGVSQAN uniprot.org/ NDPHLVPSARKAFNASGLDTAANIEGTGKKGRITSEDVKKAVASVNKPQQQTVVINQGARYEKRVK uniprot/Q5NIJ0) MTRLRQTIANALVEVQHTNAILTTFNEVDMSAVMELANKYKDMFVKEHDTKLGEMSFFIKAATEAL KKEPDVNASIDGDEIVYHNYFDIGIAVGTDRGLVVPVLADTDTKSLAELEADVLDKAIKGRDGKLS LEDMQGGTFTITNGGTYGSMLSTPIINSPQSAILGMHNIVERPVVVKGEIKIRPIMYLALSYDHRI IDGGTSVRFLKMIKELIEDPNRILLQV FTT_0583 MMALKSIVIATTVLLGSATASIAAGSDNIDTLANTNSATTQSSGFAANNFIAPFANTYSALTNKDN 88 (FopA) TWGPQDRTGQWYLGVDANGLAGTPNSPSGAGANFTIGYNINKYFAVQYNQLVGRVFAGLGEGVVNF (https://www. SNNTMFTPYAAGGAGWANLAGQATGAWDVGGGLKFELSRNVQASVDYRYIQTMAPSNISGANGRAG uniprot.org/ TNMIGAGLTWFFGGKDTTNNDTGNIQDNGATTAAQTVAMPTIDESKYVLPAGIKQCEGNFNLTEDG uniprot/Q5NH85) VACYTINGDDVTVYLDTKFAYDKATLNAKGKKAIASFVNFIKDSNISSVTVKGYASQGQTGSEFDI YNQKLSEKRAQAVADYMKQLGLDSEKIITKGFGYNDTLGGIHKSDPRNQRVEASVSAPLKEAN FTT_0901 MKKIIELSLLSLSIAGLASCSTLGLGGSDDAKASAKDTAAAQTATTEQAAAVSKPTAKVSLNKLGQ 10 (LpnA/Tu14) DKIKATVYTTYNNNPQGSVALQWQAPEGSKCHDTSFPITKYAEKNDKTWATVTVKQGNNFCSGKWT (https://www. ANVVYDKEVIASDSINI uniprot.org/ uniprot/Q5NGE4) FTT_1416c MKGLKAKIYIIFLAAVLAVISGCATDKGTQYKDGYYITTLNYNENTVYNATLQAIQNGQTFDYKSN 14 (Lpp3/ PYDISVNKNNGTDAEIVSASDSDSTDSLQVAMKKLPNNATRISIKYGSQGNSIRSSALIGIIEGNI hypothetical RYANT lipoprotein) (https://www. uniprot.org/ uniprot/Q5NF33) FTT_0890c MKKKMQKGFSLVELMVVIAIIAILAAVAIPMYSNYTTRAQLGSDLSALGGAKATVAERIANNNGDA 16 (Type IV pili SQVTILQANAAANGLPSGASVAAGTISYPSTVSGATIQLAPTVSSGAITWTCNISGVSASQVPSNC fiber building NAI block protein) (https://www. uniprot.org/ uniprot/Q5NGF5) FTT_1747 MFYKLNKQGRKIMKKTILGAMIAGGLMVSATTAMAGGVGFANVQDIFETSPLGKAKVTADEKKLKP 18 (FcpB outer QMDQLKQNITALQEKVNAYTQEKDDVQTDDSKGDTKDAQSADKVENQDKQQAQADLEKAMKDYQNL membrane MNQVQKMASDDADAFKDALTKASAQVAKEKQLDAILPAEMSLYNVDSIDVTKDVIAKMQ protein) (https://www. uniprot.org/ uniprot/Q5NEA1) FTT_1676 MEFSMANIFKKILLILLLSVIIASCGMLSNDQFVYLGHGENSPDYQLYYDKTQKLEVLIDKRNGCF 20 (Hypothetical QKDDIGICLAFTLKQAREFREYVLAKMIEIDVALAKDDYGNYAIEELRKAGVTIVNKPIKTKKVFA membrane TPVKQIVLDRKQQYHLVRKEYEIDSNLVAMVVNDKEGKKRIKVAYTVDFPGLEKEYSTELRPFIID protein) PEYLYTHMTIDAVHEAQFMQRDVMKSQINVKKKVDDYLKDVVDSDKKDIGYTHQEVASSVAALDND (https://www. LITKADEQREDKETTLASGSSISTIAKKPINQESSGSTIQTATKTASTIQVDSNDTPKTTLASDNK uniprot.org/ uniprot/Q5NEG0) FTT_1441 MLIIMIRVLNNGDNNMELQLENKQEIIDQLNKILELEMSGVVRYTHYSLMIIGHNRIPIVSWMQSQ 22 (Hypothetical ASESLTHATAAGEMITHFGEHPSLKIADLNETYQHNINDILIESLEHEKKAVSAYYELLKLVNGKS protein) IILEEYARKLIVEEETHIGEVEKMLRKPA (https://www. uniprot.org/ uniprot/Q5NF13) FTT_1778c MKLAKVLIAILLGASALSLSSCWLLVGAAVGGGTAAYISGEYSMNMSGSVKDIYNATLKAVQSNDD 24 (Hypothetical FVITKKSITSVDAVVDGSIKVDSTSFYVKIEKLIDNASKVTIKFGTFGDQAMSATLMDQIQKNL protein) (https://www. uniprot.org/ uniprot/Q5NE75) FTT_0814c MMDFKDQYNISSDGKLYINAKIMSHSSATANVIVVLQDQNGNQVYRKTDIQISPMDTYDLALAIDN 25 (Hypothetical IKAGDYKLIISSELAGAEAWQKDMNIKAVSSETSDDNNNPPPANQDINVNIESNIPYYQVNPNDSV protein) TARAWASSLGNINLANNQVIIIAWNKTLQASGNDSSAYVKCPLPKNNQISITYLVSGDLNNVNCTI (https://www. K uniprot.org/ uniprot/Q5NGM1) FTT_1043 MKLKKIVAVISCSLLGLAMSSCSINTDSVDSTNAQQVIKNTEAKGVAINKSAQDNPTIKMGANASY 26 (Mip/Peptidyl- VVGYQVGAGIAKQDFGLYDKQTIAGFADAINGNKPRISESQIRRNMETLKDKMIKKQLDTANLNKT prolyl cis- KSQEFMAQIAKMDNAIKVDDGVYYQMIKQGDGKNPKSDSQVTIAYKGTTPVIAYEDDKSKLNEVKE trans AKLIGPTEDSSDSATFPLANLIECWKDAIPQIPNGSTIILYCSPDKAYGTRAPAVIGPNQALSFEI isomerase) TLKDFK (https://www. uniprot.org/ uniprot/Q5NG16) FTT_0438 MSKHIHILGICGTFMGSLAVLAKQKGYKVIGSDLNVYPPMSTYLESQGIEILQGFDCDQLDINPDE 27 (Mpl/UDP-N- IIIGNIMKRGMPIIEKILAEKLNYFSGPEWLYQNILKYKKVIAIAGTHGKITITTMTIKILEQAGL acetylmuramate- NPSFLVGGVSSDFGVSSRYTDSEYFVIEADEYDTAFFDKRSKLIHYDPSIFVINNIEFDHADIFKD -L-alanyl- IDAIFWQFHQLLRKMPSTAKIIYNAKDDNVQKIISMGCWSELVKVNSDLGISITKHILDYSKFELC gamma-D- DINGNSVEVSWGLIGEHNALNAMSAYAVAKQLNISDEMVKDALESERGVKARLEVLSHQDNVTLYD glutamyl- DFAHHPTSIKLTLEAVANKAKDAYVVALIDPRSNIMRQGDNKDNLPMSIIEADRVLLYNHNLLKWD meso-2, 6- AKEVLKNSNNVDFIADVDDFVDCVDKLLIKHQDRNIQLVMMSNGSFDGLREKLVKLLEIK diamincheptan dioate ligase) (https://www. uniprot.org/ uniprot/Q5NHL4) FTT_0071 MEVMLMSKYAILKYADKNIEIELPVYSPSLGNDCIDVSSLVKHGIFTYDPGFMSTAACESKITYID 28 (GltA/Citrate GGKGVLLHRGYPIEEWTQKSNYRTLCYALIYGELPTDEQVKSFRQEIINKMPVCEHVKAAIAAMPQ synthase) HTHPMSSLIAGVNVLAAEHIHNGQKESQDEVAKNIVAKIATIAAMAYRHNHGKKFLEPKMEYGYAE (https://www. NFLYMMFADDESYKPDELHIKAMDTIFMLHADHEQNASTSTVALSGSTGNSPYAAIIAGITALWGP uniprot.org/ AHGGANEAVLKMLSEIGSTENIDKYIAKAKDKDDPERLMGFGHAVYKNIDPRATAMKKNCEEILAK uniprot/Q5NIJ6) LGHSDNPLLTVAKKLEEIALQDEFFIERKLFSNVDFYSGIILKAMGIPEDMFTAIFALARTSGWIS QWIEMVNDPAQKIGRPRQLYTGATNRNF FTT_0289c MKKIKLLAAYTLATLILVSCSNNASLSNDLNSAGDSIVRFVNGTFYAEIYNTSLQSVYNATLLALN 29 (Hypothetical NSNIYSVKNNTINSKDAEITGIYATDKNFFNKSGQDDFAIRLVKGNQDTINLFIKIGKLGDKQASV lipoprotein) DLLAKIQTNLGI (https://www. uniprot.org/ uniprot/Q5NI03)

The above sequences and additional sequences of protein antigens according to the present disclosure in different Francisella species and/or strains, are identifiable from sequences available in public databases such as Uniprot database (https://www.uniprot.org/uniprot/) (see e.g. Annotation\in the Tables 1 and 2) at the date of filing of the present disclosure as will be understood by a person of ordinary skill in the art.

In some embodiments, the protein antigen from the Francisella bacterium comprises DnaK, OmpA, SucB and LpnA (Tu14) preferably on a Tobacco Mosaic Virus (TMV) as an antigen carrier as such combination from Francisella Schu S4 provided complete protection in mice when used as a vaccine [18].

In some embodiments, the protein antigen from the Francisella bacterium can comprise DnaK and Tul4 in combination in particular in embodiments wherein the Francisella bacterium is Schu S4 (see Ashketar et al. [19])

In some embodiments, the protein antigen from the Francisella bacterium comprises immunogenic proteins FopA, LpnA, Lpp3 and Type IV pilus fiber, in particular in embodiments wherein the Francisella bacterium is Schu S4 (see [20].)

In some embodiments, the protein antigen from the Francisella bacterium comprises individual IglC, Ft proteins (FTT-1747 (FopB), FTT-1676, FTT-1441, or FTT-1778c) in particular in embodiments wherein the Francisella bacterium is SchuS4 in view of data showing that such combination of protein antigens induced a strong CMI response and provided, significant, albeit partial, protection against an aerosol challenge with Ft SCHU S4.

In some embodiments, the protein antigen from the Francisella bacterium, can comprise DnaK, OmpA, SucB LpnA (Tu14), FopA, Lpp3 and Type IV pilus fiber, IglC, and Ft proteins (FTT-1747 (FopB), FTT-1676, FTT-1441, and/or FTT-1778c either individually or in any combination.

In some embodiments the Francisella bacterium is Francisella tularensis, and the protein antigen from Francisella tularensis comprises intracellular growth locus protein C or IglC.

The term “intracellular growth locus protein C” or “IglC” as used herein indicates a 23-kDa protein encoded by the igl ABCD operon in the Francisella Pathogenicity Island (FPI) in the genome of a Francisella bacterium. IglC is a (3-sandwich plate comprising thirteen β sheets with approximate dimensions of 50×50×25 Å³. The β-sandwich is augmented by two α-helices and three 3₁₀-helices around its periphery. One layer of the β-sandwich is composed of six antiparallel β-strands with a strand order of β1/β2-β4-β5-β11-β10. The other layer is composed of three antiparallel b-strands: β3-β6-β9,β7-β8, and β12/β13. The two layers of the β-sandwich are packed together by virtue of extensive hydrophobic interactions at their interface. IglC is one of the most upregulated F. tularensis proteins during transcellular infection of macrophages, and is required for intracellular survival, replication and phagosome escape of F. tularensis. Studies on IglC mutants in F. tularensis subsp. novicida, holarctica, and tularensis demonstrated that IglC is essential for virulence in mice. A schematic structure of an exemplary IglC is shown in FIG. 22 which illustrates a stereoview of the crystal structure of an IglC. The 13 β-strands are numbered, and the helices are labeled A-E (including α-helices A and C and 3₁₀-helices B, D, and E).

In some embodiments, the IglC protein antigen of an antigenic combination herein described has a protein sequence of SEQ ID NO:1 shown in Table 1. SEQ ID NO:1 corresponding to an intracellular growth locus subunit C from Francisella tularensis subsp. tularensis SCHU S4.

In some embodiments, the antigenic combination herein described can comprise a Francisella protein antigen component comprising one or more derivatives of a protein antigen of a Francisella bacterium capable of inducing a cell mediated immune response in the individual.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a gene sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 or a protein sequence of SEQ ID NOs: 2, 4, 6, 8, 88, 10, 14, 16, 18, 20, 22, 24-29. In some embodiments, the Francisella protein antigen component comprises one or more protein antigens having at least 30% identity with the Francisella protein antigen having SEQ ID NOs: 1-11, 88, and 13-29 and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens. In some embodiments, the Francisella protein antigen component comprises one or more protein antigens having at least 80% identity with the Francisella protein antigen having SEQ ID NOs: 1-11, 88, and 13-29.

The term “percent identity” refers to a quantitative measurement of the similarity between sequences of a polypeptide or a polynucleotide and, in particular, indicates the amount of characters that match between two different sequences. Commonly used similarity searching programs, like BLAST, PSI-BLAST [21], SSEARCH [22, 23] FASTA [24] and the HMMER [25] can produce accurate statistical estimates, ensuring that protein sequences that share significant similarity also have similar structures.

The identity between sequences is typically measured by a process that comprises the steps of aligning the two polypeptide or polynucleotide sequences to form aligned sequences, then detecting the number of matched characters, i.e. characters similar or identical between the two aligned sequences and calculating the total number of matched characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity result is expressed as a percentage of identity.

In some embodiments, the Francisella protein antigen component comprises one or more protein antigens having a particular BLAST score with respect to the Francisella protein antigen having SEQ ID NOs: 2, 4, 6, 8, 88, 10, 14, 16, 18, 20, 22, 24-29 and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

The term “BLAST” or “Basic Local Alignment Search Tool” is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of proteins or the nucleotides of DNA sequences. A BLAST search enables a researcher to compare a query sequence with a library or database of sequences, and identify library sequences that resemble the query sequence above a certain threshold. Accordingly, BLAST or Basis Local Alignment Search Tool uses statistical methods to compare a DNA or protein input sequence, also referred to as a query sequence to a database of nucleotide and protein (subject sequences) and returns sequences hits that have a level of similarity to the query sequence ranked based on the score.

The term “score” in the context of sequence alignments, indicates a numerical value that describes the overall quality of an alignment. Higher scores correspond to higher similarity and lower scores correspond to lower similarity. The score scale depends on the scoring system used for conducting the sequence alignment.

A BLAST score, also referred to as bit score or max score in the BLAST output is a normalized score with respect to the scoring system provided by the BLAST algorithm. The BLAST score defines the highest alignment score of a set of aligned segments from the same subject (database) sequences. The score is calculated from the sum of the match rewards and the mismatch, gap open an extend penalties independently for each segment. The BLAST score normally gives the same sorting order as the expect value (E value) in the BLAST alignment output.

A BLAST score can be obtained using BLAST software suite at the NCBI website and related references and in particular at the website

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome at the date of filing of the present disclosure, as will be understood to a person skilled in the art.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 112 and 427 with the Francisella protein antigen having SEQ ID NO: 2, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 1294 and 5740 with the Francisella protein antigen having SEQ ID NO: 4, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 192 and 846 with the Francisella protein antigen having SEQ ID NO: 6, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 112 and 427 with the Francisella protein antigen having SEQ ID NO: 8, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 104 and 795 with the Francisella protein antigen having SEQ ID NO: 88, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 36.2 and 302 with the Francisella protein antigen having SEQ ID NO: 10, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 38.1 and 273 with the Francisella protein antigen having SEQ ID NO: 14, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 37.7 and 264 with the Francisella protein antigen having SEQ ID NO: 16, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 79 and 375 with the Francisella protein antigen having SEQ ID NO: 18, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 59.7 and 678 with the Francisella protein antigen having SEQ ID NO: 20, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 122 and 323 with the Francisella protein antigen having SEQ ID NO: 22, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 38.1 and 256 with the Francisella protein antigen having SEQ ID NO: 24, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 69.3 and 399 with the Francisella protein antigen having SEQ ID NO: 25, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 42 and 551 with the Francisella protein antigen having SEQ ID NO: 26, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 42.4 and 941 with the Francisella protein antigen having SEQ ID NO: 27, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 95.1 and 889 with the Francisella protein antigen having SEQ ID NO: 28, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some of these embodiments, the Francisella protein antigen component comprises one or more protein antigens having a BLAST score in a range between 37.7 and 281 with the Francisella protein antigen having SEQ ID NO: 29, and shows ability to trigger a cell-mediated immune response at a same, reduced or increased level compared to the original Francisella protein antigens.

In some embodiments of the antigenic combination herein described a Francisella protein antigen component comprises one or more derivatives of a Francisella protein antigen herein. The derivatives of a Francisella protein antigen can be an immunogenic fragment of a Francisella protein antigen with retained immunogenicity. In particular, a derivative of a Francisella protein antigen comprises an epitope capable of eliciting a same, lower or higher cell-mediated immune response.

Accordingly, a derivative of a protein antigen having any one of SEQ ID NOs: 1-11, 88, and 13-29 or a sequence with at least 30% identity thereof is a fragment of said protein antigen comprising one or more epitopes of the antigen capable of eliciting a cell-mediated immune response in an individual. In general, an epitope, when discussed in connection with proteins refers to the shortest amino acid sequence in a protein that maintains stimulatory capacity for T cells. T cell epitopes of a protein antigen can be presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules. In humans, antigen-presenting cells are specialized to present MHC class II peptides, whereas most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length, and non-classical MHC molecules also present non-peptidic epitopes such as glycolipids.

A possible approach for identifying epitopes of an antigen, is that of synthesizing a complete panel of peptides, with a given length and a predetermined overlapping sequence, such that it encompasses an entire protein antigen. Once peptide panels have been produced, T cells can be tested with different methods to determine the stimulatory immunodominant peptides using assays that predict T-cell immunity. Using such assays, a functional T-cell response to stimulatory peptides can be demonstrated by upregulation of activation markers, cytokine synthesis, T-cell proliferation, cytolytic, and helper T cell function as described in Pira et al., 2010 [26]. Exemplary methods for T epitope mapping include WIC-peptide multimers, solid phase WIC-peptide complexes, lymphoproliferation, activation markers induced on specific T cells, ELlspot, intracytoplasmic cytokine staining (ICS), cytokine secretion and cell surface capture, cytokine secretion and well surface capture (cell-ELISA) and others identifiable by a skilled person. For example, several activation markers have been used for identification, enumeration, and selection of antigen specific CD4 and CD8 T cells. These markers for antigen activated T cells include CD137 for detection of CD8 cells and CD154 for detection of CD4 cells. Coexpression of CD25 and CD134 also identifies antigen specific CD4 T cells. Therefore, the same markers can also be applied for epitope mapping. In addition, T cells that secrete a given cytokines as a consequence of antigen recognition on APC can be detected by ELlspot as will be understood by a skilled person.

In some embodiments, synthesizing a complete panel of peptides, with a given length and a predetermined overlapping sequence, such that it encompasses an entire protein antigen can be performed by dividing the original protein antigen into protein antigen overlapping peptides with preset peptide length and offset number (the number of overlapping amino acids between two adjacent peptides) the protein antigen overlapping peptides having a sequence such that the protein antigen overlapping peptides encompass the entire protein antigen sequence. The offset number reflects the degree of overlapping. In general, the peptide length is selected between 8 to 20 amino acids and the offset number can be from 1 to 7.

In some embodiment, the protein antigen overlapping peptides can be designed to be 15 amino acids in length and sequentially shifted in sequence by 5 residues. For optimization experiments, peptides can be designed with a shorter length, such as 9 amino acids, and shifted in sequence by 1 residue (see Example 21 wherein the Francisella protein antigen is IglC). In some embodiments the protein antigen overlapping peptides can be designed to have a sequence covering only a portion of the protein antigen as will be understood by a skilled person.

A library of protein antigen overlapping peptides can then be generated for example by solid phase peptide synthesis on a solid support such as a resin, as well as with other techniques identifiable by a skilled person and the synthesized protein antigen overlapping peptides can then be tested for their immunogenicity as will be understood by a skilled person (see Example 22). Additional peptides can be designed and tested in view of the results of the immunogenicity tests (see e.g. the fine mapping of immunogenic peptide 9 in Example 22). In general, even in presence of negative results in a round of immunogenicity tests the process of designing and synthesizing peptide library can be repeated until epitopes of a protein antigen are identified.

Exemplary peptides from Francisella protein antigen IglC of SEQ ID NO: 2 comprise immunogenic T cell epitopes, TSGETIHVRTDPTAC (SEQ ID NO: 33), GSHPNCRLFIDSLTI (SEQ ID NO: 37) NCRLFIDSLTIAGEK (SEQ ID NO: 38), VTKADSATAAASVIR (SEQ ID NO: 45), DSATAAASVIRLSIT (SEQ ID NO: 46), YPISAKAFSISIEPT (SEQ ID NO: 67), AKAFSISIEPTELMG (SEQ ID NO: 68), SKDGMRYHIISIDGL (SEQ ID NO: 72), MRYHIISIDGLTTSQ (SEQ ID NO: 73). In some embodiments, the exemplary peptides from Francisella protein antigen IglC of SEQ ID NO: 2 comprise NCRLFIDSL (SEQ ID NO: 81).

Peptides comprising immunogenic epitopes of additional protein antigens herein described, such as protein antigens having SEQ ID NO:4, 6, 8, 88, 10, 14, 16, 18, 20, 22, and 24 to SEQ ID NO: 29, can be identified with the above approach as will be understood by a skilled person.

Francisella protein antigens herein described comprise protein antigens obtained from recombinant protein expression as will be understood by a person of ordinary skill in the art.

Recombinant Francisella protein antigen can be created artificially using recombinant DNA technology identifiable by a person skilled in the art of molecular biology. In general, polynucleotides encoding Francisella protein antigens can be cloned using commercially available reagents from vendors such as Qiagen, Invitrogen, Applied Biosystems, Promega, and others, following standard molecular biology methods known in the art, such as those described in Sambrook and Russell (2001) Molecular Cloning, A Laboratory Manual. Synthetic DNA. Genomic DNA or cDNA encoding pyocyanin demethylase derivatives can be cloned into an expression vector. Expression vectors can comprise plasmid DNA, viral vectors, or non-viral vectors, among others known to those skilled in the art, comprising appropriate regulatory elements such as promoters, enhancers, and post-transcriptional and posttranslational regulatory sequences, as would be understood by a skilled person. Promoters can be constitutively active or inducible. RNA can be isolated from a cell, such as Francisella tularensis and cDNA produced by reverse transcription using standard techniques and commercial kits. Alternatively, genomic DNA can be purified from the cell, and cDNA or genomic DNA encoding one or more pyocyanin demethylases isolated, following methods known to those in the art. PCR-based amplification of the gene of interest can be performed using appropriately designed primer pairs (e.g. using PrimerDesign or other programs known to those skilled in the art). An encoded tag can be incorporated into the primer design (e.g. encoding a His-tag designed to be fused to the N- or C-terminus of the recombinant enzyme) to facilitate protein purification (e.g. using commercially-available His-tagged protein purification columns/kits), as described below. PCR-based amplification can be followed by ligation (e.g. using T4 DNA ligase) of the amplicon into an appropriate expression cassette in a plasmid suitable for propagation in bacteria or other cells, such as transformation-competent E. coli, followed by growth of transformed cell cultures, purification of the plasmid for confirmation of the cloned pyocyanin demethylase by DNA sequence analysis, among other methods known to those skilled in the art.

Cloned recombinant Francisella protein antigens can be expressed using cell-based methods or cell-free methods, following standard techniques and using commercially available kits. Cell-based methods for expression of recombinant Francisella protein antigens can include expression in prokaryotic or eukaryotic cell cultures, such as E. coli or other bacterial cells, yeast strains, insect cells, or mammalian cells, among other known to those skilled in the art.

An antigenic Francisella protein, an antigenic Francisella polysaccharide and/or derivative thereof, either experimentally identified or computationally predicted, can be assessed for immunogenicity and protective capacity in a suitable animal model using approaches known to a person skilled in the art. Murine model systems can be employed where animals are vaccinated with the antigen of interest and the humoral and cell mediated immune response to vaccination is characterized. In the case of humoral responses, serum from vaccinated animals can be screened for the presence of antigen specific antibodies using traditional ELISA methodologies. Cell mediated immunity can be assessed using recall responses to quantify the ability of recombinant protein to elicit activation of T cells in vaccinated animals. Protection can then be assessed in vaccinated animals that are subsequently challenged with a lethal dose of Francisella and assessed for survival.

In some embodiments, the antigenic combination comprising a Francisella protein antigen from a specific Francisella bacterium or a protein antigen having at least 30% identity with the Francisella protein antigen from a specific Francisella bacterium, together with an antigenic Francisella polysaccharide has protective effects against the specific Francisella bacterium itself.

In some embodiments, the antigenic combination comprising a Francisella protein antigen from a specific Francisella bacterium together with an antigenic Francisella polysaccharide is expected to have a protective effect against a different Francisella bacterium which has a protein antigen having at least 30% identity with respect to the Francisella protein antigen from a specific Francisella bacterium.

In some embodiments, the target Francisella bacteria is a human pathogen selected from subspecies tularensis, subspecies holarctica and novicida.

In some embodiments, the polysaccharide of the antigenic combination is from the target Francisella bacteria. In some embodiments, the polysaccharide of the antigenic combination is from a Francisella bacteria other than the target Francisella bacteria. the polysaccharide of the antigenic combination is synthetic.

In some embodiments, the antigenic combination comprising a Francisella protein antigen from a Francisella tularensis subspecies tularensis together with an antigenic Francisella polysaccharide provides protection effects against a Francisella novicida F6168 bacterium which has a protein antigen having at least 71% identity with respect to the Francisella protein antigen from the Francisella tularensis subspecies tularensis.

In some other embodiments, the antigenic combination comprising a Francisella protein antigen from a Francisella tularensis subspecies tularensis together with an antigenic Francisella polysaccharide provides protection effects against a Francisella holarctica bacterium which has a protein antigen having at least 90% identity with respect to the Francisella protein antigen from the Francisella tularensis subspecies tularensis.

In antigenic combination herein described, at least one antigenic Francisella polysaccharide (LPS and/or CPS) and at least one protein antigen from a Francisella bacterium are comprised together with at least one adjuvant.

The term “adjuvant” as used herein indicates an agent that stimulates the immune system but that is not antigenic in itself. Typically, adjuvants are used in connection with antigens and/or vaccine composition to increase the response to one or more antigen of choice. Adjuvants can be added to an antigenic combination to modify the immune response by boosting it such as to give a higher amount of antibodies and a longer-lasting protection, thus minimizing the amount of injected foreign material. Adjuvants can also be added to an antigenic combination to enhance the efficacy of the related antigens by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine. Adjuvants are also used in the production of antibodies from immunized animals. There are different classes of adjuvants that can push immune response in different directions as will be understood by a skilled person. In some embodiments, adjuvants that can be comprised in antigenic combinations herein described include naturally occurring hydrophobic or amphipathic adjuvants, immunostimulatory peptides (e.g. f-Met-Leu-Phe), muramyl dipeptide (including the analog muroctasin), saponins (e.g. gylcosidic terpenes from Qillaja saponaria), toxins (e.g. tetanus, cholera), oligonucleotide CpG motifs (e.g. Agatolimod), organic compounds (e.g. squalene, soribitol oleate esters), alpha-galactosyl ceramide, and lipotichoic acid (LTA), or hydrophilic adjuvants synthetically appended with a hydrophobic moiety, including microbial derivatives (e.g. muramyl dipeptide (MDP), flagellin), plant derivatives (e.g. saponins), and immunostimulatory proteins (e.g. cytokines, chemokines, flagellin, toxins, and derivative peptides), immunostimulatory carbohydrates and polysaccharides, and immunostimulatory nucleic acids (e.g. cholesterol-tagged CpG oligonucleotides).

Additional adjuvant suitable to be used in antigenic combination in the sense of the current disclosure include AddaVax™, Adju-Phos® adjuvant, Alhydrogel® adjuvant 2%, CFA, IFA, Quil-A® adjuvant, 2′3′-cGAMP VacciGrade™, 3′3′-cGAMP VacciGrade™, c-di-AMP VacciGrade™, 2′3′-c-di-AM(PS)2 (Rp,Rp) VacciGrade™, c-di-GMP VacciGrade™, Chitosan VacciGrade™, CL401 VacciGrade™, CL413 VacciGrade™, CL429 VacciGrade™, Flagellin FliC VacciGrade™, Gardiquimod VacciGrade™, Imiquimod VacciGrade™, LPS-EB VacciGrade™, MPLA-SM VacciGrade™, MPLA Synthetic VacciGrade™, N-Glycolyl-MDP VacciGrade™, ODN 1585 VacciGrade™, ODN 1826 VacciGrade™, ODN 2006 Vaccigrade™ ODN 2395 VacciGrade™, Pam3CSK4 VacciGrade™, Poly(I:C) (BMW) VacciGrade™, R848 VacciGrade™, TDB VacciGrade™, and others identifiable to a person skilled in the art.

The antigenic combination herein described can comprise an antigenic Francisella polysaccharide (e.g. LPS and/or CPS, and/or one or more derivatives thereof) in an amount from about 0.1 μg antigen/vaccination to about 100 μg antigen/vaccination, preferable 2-20 μg antigen/vaccination. The antigenic combination also comprises at least a Francisella protein antigen, each protein antigen in an amount from about 0.1 μg antigen/vaccination to about 100 μg antigen/vaccination, preferably 5-50 μg antigen/vaccination. The antigenic combination also comprises an adjuvant in an amount from about 0.001 μg to 200 μg, preferably between 0.5 and 50 μg.

A variety of factors will affect the dose of the antigenic Francisella polysaccharide, the Francisella protein antigen and the adjuvant, including the type of adjuvant and antigens being used, the administration route, the animal model used for testing, and whether the adjuvant is conjugated to the NLPs as will be understood by a person skilled in the art. Methods for dose conversion from animal model to humans and estimation of starting does for clinical trials can be found in related references such as Nair, A. B et al., [27]as will be understood by the skilled person.

Optimal vaccine composition and immunization regimen can be determined empirically by a skilled person. The general approach is to select and modify one parameter of the vaccine composition or immunization regimen while keeping all other parameters unchanged. The effect of the modification can be evaluated by immunizing separate groups of animals with the unmodified and modified vaccines and then challenging the vaccinated animals with a lethal dose of aerosolized Francisella tularensis SCHU S4 strain. The stringency of the test can be increased with larger challenge doses. Modifications that significantly improve survival, bacterial burden, or other measurable physiological parameters such as body weight, temperature, etc. compared to the unmodified vaccine will be selected for further development. Similar approaches can be used to optimize other parameters and to optimize the vaccine composition or immunization regimen for other animal species, including humans.

In particular, the antigenic combination herein described can comprise the Francisella LPS and Francisella protein antigen at a ratio between 1:0.1 and 1:10 by mass. In particular, the ratio between Francisella antigens and adjuvants herein described can range from 1:0.00001 and 1:2000 by mass. In some embodiments, the LPS to protein antigen ratio is 1:4 by mass. In some embodiments, the immunogenic composition comprises about 20 μg of protein antigen and about 5 μg of LPS (see Examples 12-17).

In embodiments herein described, the antigenic combination can comprise more than one protein antigen or derivative thereof, included in an antigenic combination together with one or more Francisella LPS and one or more adjuvants. In some embodiments, up to five different protein antigens can be included in the antigenic combination herein described. In some preferred embodiments, the antigenic combination comprises one LPS and one protein antigen, from the Francisella bacterium. For example in some embodiments an antigenic component of an antigenic combination herein described can comprise peptides of SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73 alone or in combination one with the other and/or with protein antigens such as IglC, SucB, OmpA, and DnaK in Table 1 or derivatives thereof in combination with any one of the adjuvants of the disclosure and preferably also in combination with one or more Francisella LPS.

In some embodiments, the antigenic combination herein described can further comprise one or more additional immunogenic agents. The term “immunological agent” as used herein indicates a compound that is able to interfere with the immune system of an individual, and in particular provoke, reduce, enhance or impair a response of the immune system under same or comparable conditions. Exemplary immunological agents comprise antigen and adjuvants.

In some embodiments herein described, the components of the antigenic combination herein described can be presented on a carrier.

A “carrier' in the sense of the disclosure indicates a substrate used in the process of delivery of an active principle which serves to improve the selectivity, effectiveness, and/or safety of the administration of the active principle. Carriers in the sense of the disclosure can encompass substrates selected to control the release of the active principle into systemic circulation of an individual, for example by slow release of the active principle over a long period of time (typically diffusion) or by triggered release at the active principle's target by some stimulus, such as changes in pH, application of heat, and activation by light. Carriers in the sense of the disclosure encompass substrates selected to improve the pharmacokinetic properties, specifically the bioavailability, of an active principle with poor water solubility and/or membrane permeability. Exemplary carriers in the sense of the disclosure include liposomes, polymeric micelles, microspheres, and nanoparticles. Methods and techniques to attach an active principle to a carrier are identifiable by a skilled person and comprise adsorption, integration into the bulk structure, encapsulation, and covalent bonding. Different types of carriers utilize different methods of attachment, and some carriers can even implement a variety of attachment methods as will be understood by a skilled person.

In some embodiments, at least one of the antigenic polysaccharide from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium, and the adjuvant can be presented on a single or on separate carriers. In particular, in some of these embodiments, at least two of the protein antigens from the Francisella bacterium, the antigenic polysaccharide from the Francisella bacterium or derivative thereof and the adjuvant can be jointly presented on a single carrier which can be of a same or different type. In some embodiments the protein antigen from the Francisella bacterium, the antigenic polysaccharide from the Francisella bacterium or derivative thereof and the adjuvant can be presented on separate carriers.

In some embodiments, the antigenic polysaccharide, protein antigens, and/or adjuvants comprised in the antigenic combination herein described can be carried by a membrane-containing platform configured to provide a controllable and stable environment for the antigens to be incorporated while preserving their native conformations. The membrane-containing platform can also allow co-localized presentation of the antigens and/or adjuvants thus resulting in enhanced immunogenic response. Membrane containing platform can be isolated from existing membrane (e.g. cell membranes) or assembled from a number of components in vitro. Examples of membrane-containing platform include liposomes, micelles, membrane bilayers, nanolipoprotein particles and others identifiable by a person skilled in the art. In some embodiments the carrier can be provided by a nanolipoprotein particle.

In some embodiments, the antigenic polysaccharide from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium, and/or the adjuvant can comprise a hydrophobic region configured to attach to a membrane lipid bilayer of a membrane derived platform.

For example, membrane proteins can be detergent solubilized and incorporated into membrane-derived platform during platform formation. In another example, a hydrophilic adjuvant (e.g. CpG oligonucleotide) that appended with a hydrophobic moiety (e.g. cholesterol) can be added to an aqueous solution of pre-formed membrane-containing platform. The cholesterol moiety of the adjuvant molecule will anchor the adjuvant into the membrane bilayer.

In some embodiments, Francisella lipopolysaccharides having a lipid A component, a core polysaccharide component and an O-antigen domain can be attached to a membrane-containing platform via the interactions between hydrophobic lipid A component and a membrane lipid bilayer of a membrane derived platform. The lipid A component of the Francisella LPS can be embedded into the membrane-containing platform and the polysaccharide and the O-antigen components are presented on the surface of the membrane-containing platform. In general, LPS can be incorporated into membrane-containing platform during platform formation. For example, LPS-liposomes can be formed by rehydrating dried lipid films in an aqueous solution containing LPS. During subsequent vortexing or sonication, the LPS will embed within the formed liposomes. Generic procedure for incorporating LPS into liposomes can be found in Watanabe et al 2013 [28].

In some embodiments, one or more of the antigenic polysaccharide from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium, and/or the adjuvant, can comprise an anchor compound presented for binding with a corresponding anchor compound substrate on the carrier. In particular, in some embodiments the antigenic polysaccharide lacking a lipid A or lipid-A like moiety can be modified to present an anchor compound suitable for attachment to the anchor compound substrate of the carrier.

For example, soluble protein antigens that incorporate a poly-histidine peptide tag can be conjugated to a lipid-containing platform that features a lipid chelating nickel at the polar headgroup. The polyhistidine-tagged antigen will bind to the chelated nickel at the lipid platform surface and remain tethered. Similarly, protein adjuvants (e.g. flagellin) that incorporate a poly-histidine peptide tag can be conjugated to a lipid-containing platform that features a lipid chelating nickel at the polar headgroup.

In some embodiments, Francisella polysaccharide derivatives lacking a lipid component, Francisella protein antigens and/or adjuvants can be appended with a lipidic moiety or compound and then anchored in a membrane-derived platform. Exemplary lipidic compound include acyl chains, hydrophobic carbon chains, and others identifiable to a person skilled in the art. For example, CpG can be modified to include a cholesterol moiety. The cholesterol moiety anchors the highly-polar CpG molecule to the membrane-derived platform.

In some embodiments, Francisella polysaccharide, derivatives thereof, protein antigens, derivatives thereof, and/or adjuvants can be attached to a phospholipid functionalized with an anchor compound substrate through binding of an anchor compound attached to the Francisella polysaccharide, protein antigen and/or adjuvant and the corresponding anchor compound substrate.

In some embodiments where the antigen is a derivative of a Francisella protein antigen, the structure of the derivative can be identified using bioinformatics tools such as TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/at the date of filing of the present disclosure) which can be used to predict transmembrane helices in protein sequences in order to ascertain a mode of NLP conjugation for each antigen listed in Table 1. In particular, accordance with an exemplary approach based on such predictions, the derivative Francisella protein antigen can be binned into two groups: 1) proteins with no putative transmembrane domains, and 2) proteins with one or more transmembrane domains. This binning allows one to tailor the vaccine formulations, especially with regards to formulation into a Nanolipoprotein Particle (NLP) or other lipidic particle carrier described herein.

Based on the TMHMM predictions, seven of the proteins listed in Table 1 contain at least one TM helix. For those proteins lacking putative transmembrane domains, including FTT 1441, DnaK, OmpA and SucB, these proteins can be recombinantly expressed to incorporate a functional anchor (e.g. poly-his tag) that will be used to anchor the soluble Ft protein to the NLP surface using a cognate anchor compound on the NLP bilayer surface as described for IglC below. For those proteins with putative membrane domains, including FopA, LpnA/Tul4, Lpp3, Type IV pilus fiber building block protein, FopB, FTT_1676 and FTT_1778c, recombinantly expressed Ft protein can be incorporated during the assembly process of NLP or other lipidic platform described herein such that the transmembrane domains of the Ft protein are embedded within the lipid bilayer.

In some embodiments, the antigenic combinations herein described are incorporated into nanolipoprotein particles to provide immunogenic nanolipoprotein particles.

The term “nanolipoprotein particle” “nanodisc” “rHDL” or “NLP” as used herein indicates a supramolecular complex formed by a membrane forming lipid arranged in a lipid bilayer stabilized by a scaffold protein. The membrane forming lipids and scaffold protein are components of the NLP. In particular, the membrane forming lipid component is part of a total lipid component (herein also membrane lipid component or lipid component) of the NLP together with additional lipids such as functionalized lipids and/or lysolipids, that can further be included in the NLPs as will be understood by a skilled person upon reading of the present disclosure. The scaffold protein component is part of a protein component of the NLP together with additional proteins such as membrane proteins, target proteins and other proteins that can be further included as components of the NLPs as will be understood by a skilled person upon reading of the present disclosure. Additional components can be provided as part of the NLP herein described as will be understood by a skilled person. In particular, the membrane lipid bilayer can attach membrane proteins or other amphipathic compounds through interaction of respective hydrophobic regions with the membrane lipid bilayer. The membrane lipid bilayer can also attach proteins or other molecule through anchor compounds or functionalized lipids as will be understood by a skilled person upon reading of the disclosure. In a nanolipoprotein particle, the membrane lipid bilayer can be confined in a discoidal configuration by the scaffold protein. Predominately discoidal in shape, nanolipoprotein particles typically have diameters between 5 to 25 nm, share uniform heights between 3 to 6 nm and can be produced in yields ranging between 30 to 90%.

In particular, in embodiments herein described the nanolipoprotein particle can be formed by a lipid bilayer confined in a discoidal configuration by a scaffold protein. In this configuration, the lipid bilayer confined by the scaffold protein can be 3-6 nanometers in thickness, the nanolipoprotein particle can have an overall diameter of 5-25 nanometers, and the scaffold protein on the particle can have a thickness of 1-2 nanometers. In some embodiments, an entire NLP structure can be up to 600 kilodaltons in weight.

The particular membrane forming lipid, scaffold protein, the lipid to protein ratio, and the assembly parameters determine the size and homogeneity of nanolipoprotein particles as will be understood by a skilled person. In the nanolipoprotein particle, the membrane forming lipid are typically arranged in a membrane lipid bilayer confined by the scaffold protein in a discoidal configuration as will be understood by a skilled person.

The term “membrane forming lipid” or “amphipathic lipid” as used herein indicates a lipid possessing both hydrophilic and hydrophobic moieties that in an aqueous environment assembles into a lipid bilayer structure that consists of two opposing layers of amphipathic molecules known as polar lipids. Each polar lipid has a hydrophilic moiety, i.e. a polar group such as, a derivatized phosphate or a saccharide group, and a hydrophobic moiety, i.e., a long hydrocarbon chain. Exemplary polar lipids include phospholipids, sphingolipids, glycolipids, ether lipids, sterols, alkylphosphocholines and the like. Amphipathic lipids include but are not limited to membrane lipids, i.e. amphipathic lipids that are constituents of a biological membrane, such as phospholipids like dimyristoylphosphatidylcholine (DMPC) or dioleoylphosphoethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC), or dipalmitoylphosphatidylcholine (DPPC). In a preferred embodiment, the lipid is dioleoylphosphatidylcholine (DOPC).

The term “scaffold protein” as used herein indicates any amphipathic protein that is capable of self-assembly with amphipathic lipids in an aqueous environment, organizing the amphipathic lipids into a bilayer disc, and comprise apolipoproteins, lipophorins, derivatives thereof (such as truncated and tandemly arrayed sequences) and fragments thereof (e.g. peptide fragments and synthetic peptides) which maintains the amphipathic nature and capability of self-assembly, such as apolipoprotein E4 (22 kD fragment), lipophorin III, apolipoprotein A-1 and the like. In general, scaffold proteins have an alpha helical secondary structure in which a plurality of hydrophobic amino acids form a hydrophobic face and a plurality of hydrophilic amino acids form an opposing hydrophilic face. In some embodiments, rationally designed amphipathic peptides and synthetic apolipoproteins which maintain an amphipathic structure and capability of self-assembly can serve as a scaffold protein of the NLP.

The term “apolipoprotein” as used herein indicates an amphipathic protein that binds lipids to form lipoproteins. The term “amphipathic” pertains to a molecule containing both hydrophilic and hydrophobic properties. Exemplary amphipathic molecules comprise molecules having hydrophobic and hydrophilic regions/portions in its structure. Examples of biomolecules which are amphipathic include but not limited to phospholipids, cholesterol, glycolipids, fatty acids, bile acids, saponins, and additional lipids identifiable by a skilled person. A “lipoprotein” as used herein indicates a biomolecule assembly that contains both proteins and lipids. In particular, in lipoproteins, the protein component surrounds or solubilizes the lipid molecules enabling particle formation. Exemplary lipoproteins include the plasma lipoprotein particles classified under high-density (HDL) and low-density (LDL) lipoproteins, which enable fats and cholesterol to be carried in the blood stream, the transmembrane proteins of the mitochondrion and the chloroplast, and bacterial lipoproteins. In particular, the lipid components of lipoproteins are insoluble in water, but because of their amphipathic properties, apolipoproteins such as certain Apolipoproteins A and Apolipoproteins B and other amphipathic protein molecules can organize the lipids in a bilayer orientation with exposed hydrophilic moieties, creating the lipoprotein particle that is itself water-soluble, and can thus be carried through water-based circulation (e.g. blood, lymph in vivo or in vitro). Apolipoproteins known to provide the protein components of the lipoproteins can be divided into six classes and several sub-classes, based on the different structures and functions. Exemplary apolipoprotein known to be able to form lipoproteins comprise Apolipoproteins A (apo A-I, apo A-II, apo A-IV, and apo A-V), Apolipoproteins B (apo B48 and apo B100), Apolipoproteins C (apo C-I, apo C-II, apo and apo C-IV), Apolipoproteins D, Apolipoproteins E, and Apolipoproteins H. For example, apolipoproteins B can form low-density lipoprotein particles, and have mostly beta-sheet structure and associate with lipid droplets irreversibly, while Apolipoprotein Al comprise alpha helices and can associate with lipid droplets reversibly forming high-density lipoprotein particles.

The term “protein” as used herein indicates a polypeptide with a particular secondary and tertiary structure that can interact with another molecule and in particular, with other biomolecules including other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and/or small molecules. The term “polypeptide” as used herein indicates an organic linear, circular, or branched polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full-length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer, peptide, or oligopeptide. In particular, the terms “peptide” and “oligopeptide” usually indicate a polypeptide with less than 100 amino acid monomers. In particular, in a protein, the polypeptide provides the primary structure of the protein, wherein the term “primary structure” of a protein refers to the sequence of amino acids in the polypeptide chain covalently linked to form the polypeptide polymer. A protein “sequence” indicates the order of the amino acids that form the primary structure. Covalent bonds between amino acids within the primary structure can include peptide bonds or disulfide bonds, and additional bonds identifiable by a skilled person. Polypeptides in the sense of the present disclosure are usually composed of a linear chain of alpha-amino acid residues covalently linked by peptide bond or a synthetic covalent linkage. The two ends of the linear polypeptide chain encompassing the terminal residues and the adjacent segment are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus) based on the nature of the free group on each extremity. Unless otherwise indicated, counting of residues in a polypeptide is performed from the N-terminal end (NH₂-group), which is the end where the amino group is not involved in a peptide bond to the C-terminal end (—COOH group) which is the end where a COOH group is not involved in a peptide bond. Proteins and polypeptides can be identified by x-ray crystallography, direct sequencing, immunoprecipitation, and a variety of other methods as understood by a person skilled in the art. Proteins can be provided in vitro or in vivo by several methods identifiable by a skilled person. In some instances where the proteins are synthetic proteins in at least a portion of the polymer two or more amino acid monomers and/or analogs thereof are joined through chemically-mediated condensation of an organic acid (—COOH) and an amine (—NH₂) to form an amide bond or a “peptide” bond.

As used herein the term “amino acid”, “amino acid monomer”, or “amino acid residue” refers to organic compounds composed of amine and carboxylic acid functional groups, along with a side-chain specific to each amino acid. In particular, alpha- or α-amino acid refers to organic compounds composed of amine (—NH₂) and carboxylic acid (—COOH), and a side-chain specific to each amino acid connected to an alpha carbon. Different amino acids have different side chains and have distinctive characteristics, such as charge, polarity, aromaticity, reduction potential, hydrophobicity, and pKa. Amino acids can be covalently linked to form a polymer through peptide bonds by reactions between the amine group of a first amino acid and the carboxylic acid group of a second amino acid. Amino acid in the sense of the disclosure refers to any of the twenty naturally occurring amino acids, non-natural amino acids, and includes both D an L optical isomers.

In some embodiments, an immunogenic nanolipoprotein particle comprises and presents the antigenic combination herein described. The immunogenic nanolipoprotein particle herein described comprises: a scaffold protein, a membrane forming lipid, at least one antigenic Francisella polysaccharide (e.g. LPS and/or CPS and/or derivatives thereof in any combination), at least one protein antigen from Francisella, and an adjuvant, wherein the membrane forming lipids are arranged in a discoidal membrane lipid bilayer stabilized by the scaffold protein and the at least one Francisella polysaccharide or derivative thereof, at least one protein antigen from Francisella, and an adjuvant are attached to the discoidal membrane lipid bilayer. Accordingly, in these embodiments the NLPs herein described are provided as carriers for presenting Francisella polysaccharide, protein antigens and adjuvants.

The term “present” as used herein with reference to a compound or functional group indicates attachment performed to maintain the chemical reactivity of the compound or functional group as attached. Accordingly, a functional group presented on a molecule is able to perform under the appropriate conditions the one or more chemical reactions that chemically characterize the functional group. A compound presented on a particle is able to perform under the appropriate conditions the one or more chemical reactions that chemically characterize the compound. In embodiments, where the compound is or comprises an immunological agent, the immunological agent presented maintains the complex of reactions that are associated with the immunological activity characterizing the agent at issue. Accordingly, presentation of an immunological agent indicates attachment such that the immunological activity associated to the immunological agent attached is maintained.

The term “attach” or “attached” as used herein, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment such that for example where a first compound is directly bound to a second compound or material, and the embodiments wherein one or more intermediate compounds, and in particular molecules, are disposed between the first compound and the second compound or material.

In some embodiments, at least one of the Francisella antigenic polysaccharide, derivative thereof, protein antigen from Francisella, derivative thereof and the adjuvant, comprises a hydrophobic region and is attached to the membrane lipid bilayer through interaction of the hydrophobic region with the membrane lipid bilayer.

In particular, in some embodiments wherein the antigenic Francisella polysaccharide component comprises at least one Francisella polysaccharide and/or derivative thereof comprising a hydrophobic moiety (such as hydrophobic lipid A or lipid A-like moiety of LPS), the antigenic Francisella polysaccharide component is attached to the NLPs via the hydrophobic moiety, which anchors the polysaccharide (e.g. LPS and/or CPS) and/or the derivative thereof to the NLP. In particular, in those embodiments the polysaccharide molecules forming the antigenic polysaccharide component from the Francisella bacterium, comprise a lipid component embedded into the nanolipoprotein particle and a polysaccharide component presented on the surface of the nanolipoprotein particle. Similarly, Francisella protein antigen components comprising one or more Francisella protein antigens and/or derivative thereof having a hydrophobic region (such as LpnA/Tu14, FopA1, Lpp3, Type IV pilus fiber building block protein and hypothetical proteins in Table 1 or derivative thereof having at least one transmembrane domain) can be embedded into the NLPs via the hydrophobic transmembrane domain with the epitope domain presented on the surface of the nanolipoprotein particle.

In some embodiments, a lipid component of a Francisella polysaccharide or derivative thereof can be replaced with a lipidic moiety to anchor the polysaccharide component and/or the O-antigen to the NLP during self-assembly.

In some embodiments, a Francisella polysaccharide or derivative thereof lacking a lipid component, a Francisella protein antigen or derivative thereof and/or an adjuvant can also be appended with a lipidic moiety or compound. Exemplary lipidic compound include acyl chains, hydrophobic carbon chains, and others identifiable to a person skilled in the art. For example, a Francisella polysaccharide derivative lacking a lipid component can be appended with a hydrophobic carbon chain to anchor the O-antigen or O-antigen together with a core polysaccharide to the membrane lipid bilayer. Similarly, Francisella protein antigens that do not have a transmembrane domain (such as IglC, SucB, OmpA, and DnaK in Table 1) or derivatives thereof such as peptides of SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 45), SEQ ID NO: 46, SEQ ID NO: 67, (SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73.can be appended with a lipidic moiety or compound such as a hydrophobic carbon chain to anchor the Francisella protein antigens to the membrane lipid bilayer as will be understood by a skilled person.

In some embodiments, adjuvants having a hydrophobic region can be attached to an NLP through interaction of the hydrophobic region with the membrane lipid bilayer. Exemplary adjuvants falling in this group comprise amphipathic adjuvants such as cholesterol-tagged CpG can be added to preformed NLPs and will be anchored by the cholesterol moiety. Other adjuvants (for example Fsl-1, which is a peptide with acyl chains) can be added to preformed discs. Other adjuvants, like monophosphoryl Lipid A are incorporated during the assembly process since they are highly non-polar.

In some embodiments, the NLP can comprise a functionalized amphipathic compound and at least one of the antigenic Francisella polysaccharide, derivatives thereof, Francisella protein antigens, derivatives thereof and/or adjuvants can be attached to functionalized membrane-forming lipids through binding of an anchor compound attached to the antigenic Francisella polysaccharide, antigen and/or adjuvant and a corresponding anchor compound substrate attached to the functionalized membrane-forming lipids.

The terms “functionalize” and “functionalization” as used herein, indicates the appropriate chemical modifications of a molecular structure (including a substrate or a compound) resulting in attachment of a functional group to the molecular structure. The term “functional group” as used herein indicates specific groups of atoms within a molecular structure that are responsible for the characteristic chemical reactions of that structure. Exemplary functional groups include, hydrocarbons containing halogen groups, hydrocarbons containing oxygen groups, hydrocarbons containing nitrogen groups, hydrocarbons containing phosphorus groups and hydrocarbons containing sulfur groups, all identifiable by a skilled person.

The term “functionalized amphipathic compounds” in the sense of the disclosure indicate compound having a hydrophobic portion and a hydrophilic portion in a configuration where the hydrophobic portion anchor is capable to anchor the compound to the lipid bilayer of the NLP and the hydrophilic portion (typically consisting or comprising a hydrophilic functional group) presented on the NLP bilayer face following NLP assembly.

The use of functionalized amphipathic compounds enables attachment of various peptides or other biologics to the surfaces of the lipid of the NLP that allows some desired target features to be obtained, such as stability, affinity for a target molecule, and the like. Non-limiting examples of functional groups presented on functionalized lipids include: chelated Ni atoms, azide, anhydride, alkynes, thiols, halogens, carboxy, amino, hydroxyl, and phosphate groups, and the like.

In some embodiments, the functional group on the functionalized amphipathic compound can be a reactive chemical groups (e.g. azide, chelated nickel, alkyne, and additional reactive chemical group identifiable by a skilled person), a biologically active compound (e.g. DNA, peptide, carbohydrate, and additional biologically active group identifiable by a skilled person) or a small molecule (e.g. cellular targeting compound, adjuvant, drug, and additional small molecules identifiable by a skilled person). In some embodiments the functionalized amphipathic compound is a functionalized lipid compound. Functional groups that enhance the lipid solubility are referred to as hydrophobic or lipophilic functional groups. Functional groups that lack the ability to either ionize or form hydrogen bonds tend to impart a measure of lipid solubility to a drug molecule. The functional group can be attached to the lipid polar head through covalent or ionic bonds and “weak bonds” such as dipole-dipole interactions, the London dispersion force and hydrogen bonding, preferably covalent. Moreover, functionalization of the lipid can involve hydrophobic quantum dots embedded into the lipid bilayer. The following article is incorporated by reference in its entirety: R. A. Sperling, and W. J. Parak. “Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles”. Phil. Trans. R. Soc. A 28 Mar. 2010 vol. 368 no. 1915 1333-1383.

In some embodiments, functionalized amphipathic compounds can comprise one or more of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-((folate)amino)hexanoyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanyl), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(hexanoylamine), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanylamine), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl), 1,2-Dioleoyl-sn-Glycero-3-Phospho(Ethylene Glycol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, 1,2-di stearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000], cholesterol modified oligonucleotides, cholesterol-PEG2000-azide, cholesterol-PEG2000-Dibenzocyclooctyl, cholesterol-PEG2000-maleimide, cholesterol-PEG2000-N-hydroxy succinimide esters, cholesterol-PEG2000-thiol, cholesterol -azide, cholesterol-Dibenzocyclooctyl, cholesterol-maleimide, cholesterol-N-hydroxysuccinimide esters, cholesterol-thiol, C18 modified oligonucleotides, C18-PEG2000-azide, C18-PEG2000-Dibenzocyclooctyl, C18-PEG2000-maleimide, C18-PEG2000-N-hydroxysuccinimide esters, C18-PEG2000-thiol, C18-azide, C18-Dibenzocyclooctyl, C18-maleimide, C18-N-hydroxysuccinimide esters, C18-thiol. In some embodiments one or more functionalized amphipathic compounds are comprised together with non-functionalized membrane forming lipids in the lipid component of the NLP also comprising one or more polymerizable lipids. In some embodiments functionalized amphipathic compounds can be functionalized membrane forming lipid. In some embodiments, one or more functionalized membrane forming lipids are added or replace the membrane forming lipids in the lipid component of the NLP herein described also comprising one or more polymerizable lipids.

In several embodiments of the nanolipoprotein particle here described, the amphipathic compound is a membrane forming lipid at least a portion or all of the membrane-forming lipid is functionalized with an anchor substrate compound that is presented for binding with a target molecule. In particular, the ratio between functionalized membrane-forming lipid and membrane-forming lipids is dependent on the identity of the functionalized membrane-forming lipid, and it can be as low as 1% or even lower and as high as 100% as NLPs have been successfully formed with 100% functionalized membrane-forming lipid such as DOGS-NTA-Ni (1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)). This suggests that NLPs can be formed with any percentage of functionalized membrane-forming lipid (from 0 to 100%), depending on the specific functionalized membrane-forming lipid used.

In general, assembly of NLPs according to the present disclosure can be accomplished with a wide range of ratios of total membrane-forming lipids to scaffold proteins. For example, NLPs have been successfully formed with lipid to scaffold protein molar ratios of about 15:1 up to about 400:1. A typical assembly uses a lipid to scaffold protein molar ratio of about 100:1.

The term “anchor compound substrate” as used herein indicates a functional group capable to bind a corresponding functional group, herein also indicated as “anchor compound”, presented on another molecule, and in particular on an antigen comprising molecule or an adjuvant molecule to be attached to the nanolipoprotein particle.

The term “bind”, “binding”, “conjugation” as used herein indicates an attractive interaction between two elements which results in a stable association of the element in which the elements are in close proximity to each other. If each element is comprised in a molecule the result of binding is typically formation of a molecular complex. Attractive interactions in the sense of the present disclosure includes both non-covalent binding and, covalent binding. Non-covalent binding as used herein indicates a type of chemical bond, such as protein protein interaction, that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions. Non-covalent bonding includes ionic bonds, hydrophobic interactions, electrostatic interactions, hydrogen bonds, and dipole-dipole bonds. Electrostatic interactions include association between two oppositely charged entities. An example of an electrostatic interaction includes using a charged lipid as the functional membrane lipid and binding an oppositely charged target molecule through electrostatic interactions;

Anchor compound substrates and corresponding anchor compound capable of binding through non-covalent binding include but are not limited to those listed in Table 3 below.

TABLE 3 Non-Covalent Interactions Anchor substrate (on functionalized lipid within NLP Anchor (on a target molecule) bilayer) Poly-histidine (2-10 residues) Chelated metal cations 2-10 residue polypeptide Ni²⁺, Co²⁺, Cu²⁺, Zn²⁺ chelated on NTA, IDA Poly-arginine (5-6 residues) Negatively charged surface 5-6 residue polypeptide e.g. carboxylates, phosphates, sulfonates Proteins Biological tags Avidin (Streptavidin, neutravidin) Biotin Glutathione S-transferase (GST) Glutathione fusion proteins Strep-Tactin Strep-tag II

A covalent bond is instead a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds. In short, attraction-to-repulsion stability that forms between atoms when they share electrons is known as covalent bonding. Covalent bonding includes many kinds of interaction, including σ-bonding π-bonding, metal to non-metal bonding, agostic interactions, three-center two-electron bonds, and the like.

Anchor compound substrates and corresponding anchor compounds capable of binding through covalent binding include but are not limited to those listed in Table 4 below.

TABLE 4 Covalent Interactions Anchor (or anchor substrate) Anchor substrate (or anchor) Amine-reactive moieties Amines Active esters (e.g. succinimidyl, tetrafluorophenyl) Carbodiimide (+/− NHS)-Carboxylic acids Isothiocyanates Sulfonyl chlorides Dichlorotriazines Aryl halides Acyl azides Thiol-reactive reagents Sulfhydryls Maleimides (and derivatives) Haloacetamides (e.g. iodoacetamide) Pyridyldithio-propionate Thiosulfates Azides Acetylenes (“Click Chemistry” - formation of 1,2,3-triazol groups, ref. 7) Hydrazines/hydroxylamines/aromatic Aldehydes and ketones amines

Accordingly, exemplary functionalized membrane-forming lipids include, but are not limited to, chelated metal-bearing lipids, azide bearing lipids, maleimide bearing lipids, quaternary amine bearing lipids, carboxylate bearing lipids, propargyl bearing lipids, biotin bearing lipids, streptavidin and/or avidin bearing lipids, S-protein bearing lipids, and the like.

In some embodiments, binding or conjugation of the anchor compound can be performed by chelation. The term “chelation” as used herein indicates the binding or complexation of a bi- or multidentate ligand with a single metal ion. In particular, in some embodiments, the bi or multi-dentate ligand is part of the lipid and is capable of binding a metal ion. The ligands, which are often organic compounds, are called chelants, chelators, chelating agents, or sequestering agents. Chelating agents form multiple bonds with a single metal ion. The term “chelants” as used herein indicates a molecule that forms a stable complex with certain metal ions. Examples of chelating moieties include, but are not limited to, nitrilotriaceticacid (NTA), iminodiacetic acid (IDA), and diethylenetriamine penta-acetic acid (DTPA).

In the nanolipoprotein particles herein described, the anchor substrate compound is attached to the functionalized membrane-forming lipid so that upon assembly of the functionalized membrane-forming lipid in the nanolipoprotein particle, the anchor substrate compound is presented on the nanolipoprotein particle. Similarly, the anchor compound is attached to an immunological molecule to be presented on said NLP.

In several embodiments, the functionalized membrane-forming lipids are functionalized to present the anchor substrate compound on a hydrophilic moiety of the membrane-forming lipid to ensure presentation of the anchor substrate compound on a surface of the nanolipoprotein particle. The term “surface” as used herein indicates the exterior or upper boundary of a body or object. In particular with reference to the NLPs the term “surface” indicates they are defined by the discoidal faces. Surfaces of the NLPs form the hydrophilic portion of the NLP membrane bilayer.

Successful binding of an immunological agent to the NLP can be readily verified and quantified through a range of techniques that include but are not limited to centrifugal filtration, size exclusion chromatography, fluorescence correlation spectroscopy, cantilever-based sensing, force spectroscopy, fourier transform infrared spectroscopy, surface plasmon resonance, total internal reflection fluorescence, raman spectroscopy and additional techniques identifiable by a skilled person. In addition, binding specifically to the surface can be verified using atomic force microscopy and transmission electron microscopy and additional techniques identifiable by a skilled person.

The structure of nanolipoprotein particles herein described is illustrated in FIG. 2 which shows a schematic representation of an NLP assembly comprising a functionalized membrane-forming lipid according to some embodiments herein described.

As exemplified in the illustration of FIG. 2, panel A, purified starting components are provided that comprise a functionalized membrane-forming lipid (10), a membrane-forming lipid (20), and a scaffold protein (30). The functionalized membrane-forming lipid (10) comprises a hydrophobic moiety (11) and a hydrophilic moiety (12) and attached an anchor compound substrate (14) capable of binding to a corresponding anchor compound (41) presented on the immunological agent (40) of interest. In particular, the anchor compound substrate (14) is presented on the hydrophilic moiety (12) of the functionalized membrane-forming lipid (10). The functionalized membrane-forming lipid (10), the membrane-forming lipid (20), and the scaffold protein (30) are contacted for a time and under conditions to allow assembly of the NLP containing the functionalized membrane-forming lipid (100), shown in FIG. 2, panel A. In particular, spontaneous interaction of purified scaffold proteins, e.g. apolipoproteins, and membrane-forming lipids under appropriate conditions results in formation of nanolipoprotein particles with the nanometer-sized dimensions that are herein identified as NLPs.

In the illustration of FIG. 2, panel B, the nanolipoprotein particle (100) formed by the assembly of the functionalized membrane-forming lipid (10), the membrane-forming lipid (20) and the scaffold protein (30) is configured to present the anchor compound substrate (14) on a surface of the nanolipoprotein particle. In particular, as shown in the illustration of FIG. 2 anchor compound substrate moieties are appended to the headgroups of the functionalized membrane-forming lipid, and are presented into solution.

The amount of anchor-substrate moieties on the NLP bilayer surface can be controlled by the input ratios of membrane-forming and functionalized anchor-substrate lipids during assembly, allowing control of immunological agent loading on NLP. Generally, if 10 molar percent of the total lipid used for an assembly is a functionalized membrane-forming lipid, then approximately 10 molar percent of the total lipid within the NLP lipid bilayer will be functionalized membrane-forming lipid. This has been verified in the case of NLPs prepared with 90 molar percent DMPC and 10 molar percent of DOGS-NTA-Ni. The feed ratio to incorporated ratio of the anchor substrate moiety can further be quantitatively assesses after completion of NLP assembly using normal phase and reverse phase high performance liquid chromatography and subsequent ratios can be adjusted to achieve the desired input ratio.

Although other reagents might be added according to the desired experimental design, no other reagents have to be added to impart functionality to the NLPs since the functionalized membrane-forming lipids containing the necessary reactive group for conjugation.

As schematically shown in FIG. 2, Panel C, an immunological agent (40) bearing the appropriate corresponding anchor compound (41) (an anchor compound able to bind and accordingly be defined as complementary to the anchor-compound substrate (14)) can be attached on the NLP bilayer surface of the NLP (100).

In particular, the immunological agent (40) and the nanolipoprotein (100) are contacted for a time and under conditions to allow binding of the anchor compound (41) with the anchor compound substrate (14), which depends on the conjugation chemistry between the anchor compound and the anchor substrate compound.

Attachment of antigens and/or other immunological agents (such as adjuvants) to nanolipoprotein particles can be accomplished using any number of functionalization strategies and orientations. For example, using click chemistry, an acetylene-functionalized lipid can be conjugated with an azide-functionalized protein, or an azide-functionalized lipid can be conjugated with an acetylene-functionalized protein.

For example, attachment of antigens and/or other immunological agents (such as adjuvants) to nanolipoprotein particles can be accomplished by incorporating the molecule during NLP self-assembly. This is achieved with molecules that are hydrophobic (e.g. monophosphoryl lipid A (MPLA)) or amphipathic (e.g. LPS).

In addition, attachment of antigens and/or other immunological agents (such as adjuvants) to nanolipoprotein particles can be accomplished by adding amphipathic molecule to preformed NLP. The molecules can be inherently amphipathic (such as FSL-1) or be engineered to be amphipathic (such as CpG oligonucleotides that have been appended with a cholesterol moiety). The conjugation of azides with acetylenes (herein also indicated as click chemistry) can be achieved in buffered aqueous solution over a broad pH range for about 1 to about 24 hours to form a covalent 1, 2, 3 triazole. This reaction can be catalyzed by copper (I), typically introduced by addition of copper (II) in the presence of a reductant (e.g. ascorbic acid) to generate copper (I) in situ.

In other exemplary embodiments where attachment of a polyhistidine functionalized target molecule to a bivalent metal functionalized lipid is desired, conjugation of the poly-histidine anchor compound to the chelated metal (Ni²⁺, Co²⁻, Cu²⁺, Zn²⁺) anchor compound substrate (NTA or IDA) can be achieved over the course of an incubation ranging in time from about 5 minutes to about 2 hours at room temperature without the need of any additional components. According to this approach, no additional chelating agents (e.g. EDTA) are present in solution.

In other exemplary embodiments, where attachment of a poly-arginine functionalized immunological agent to an anionic functionalized lipid is desired, conjugation of the poly-arginine anchor compound to an anionic surface anchor compound substrate can be achieved over the course of an incubation ranging in time from about 5 minutes to about 2 hours at room temperature without the need of any additional components.

In other exemplary embodiments where attachment of an immunological agent functionalized with a protein anchor, to a lipid functionalized with a cognate biological tag is desired, conjugation of the protein anchor compounds, e.g. avidin (and derivatives such as neutravidin and the like,), glutathione S-transferase (GST) and Strep-Tactin, to the cognate biological tag anchor compound substrates biotin, glutathione, and strept-tag II, respectively, can be achieved over the course of an incubation ranging in time from about 5 minutes to about 2 hours at room temperature without the need of any additional components.

In other exemplary embodiments, where attachment of an immunological agent to a functionalized lipid is performed through conjugation of active esters to amine, conjugation of the active esters to the amines is achieved in amine-free buffered aqueous solution at a pH of about 7.0 for about 1 to about 24 hours to form a covalent amide bond. Reaction can then be quenched upon addition of free amines at neutral to basic pH. No other reagents are needed to perform conjugation in those embodiments.

In other exemplary embodiments, where attachment of an immunological agent to a functionalized lipid is performed through conjugation of carboxylic acids to amine, conjugation of the carboxylic acids to the amines can be achieved by activating the carboxylic acid to an active ester, using commercially available reagents, e.g. N-hydroxysuccinimide (NHS). This can be accomplished by combining the NHS and a dehydrating agent (e.g. carbodiimides like 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) with the target carboxylic acid. The EDC reacts with the carboxylic moiety to form a transient amine-reactive O-acylisourea, whereby NHS converts the amine-reactive O-acylisourea to an amine-reactive NETS-ester. A covalent amide bond is can then be achieved in amine-free buffered aqueous solution at a pH of 7.0 for 1 to 24 hours. Reaction can then be quenched upon addition of free amines at neutral to basic pH.

In other exemplary embodiments where attachment of an immunological agent to a functionalized lipid is performed through conjugation of isothiocyanate to amine, conjugation of the isothiocyanates to the amines is achieved in amine-free buffered aqueous solution at a pH of about 7.0 for about 1 to about 24 hours to form a covalent thiourea. Reaction can then be quenched upon addition of free amines at neutral to basic pH. According to this approach, no other reagents are needed to obtain conjugation.

In other exemplary embodiments where attachment of an immunological agent to a functionalized lipid is performed through conjugation of maleimides (and maleimide derivatives) to sulfhydryls, conjugation of the maleimides (and maleimide derivatives) to sulfhydryls can be achieved in thiol-free buffered aqueous solution at a pH between about 6.5 and about 0.5 for about 1 to about 24 hours to form a covalent thioether linkage. Maleimides can then be quenched at the completion of the reaction by the addition of free thiol. Reducing agents (e.g. tris(2-carboxyethyl)phosphine) may be used to produce free, reactive sulfhydryls, which may also be stabilized by the addition of ethylenediaminetetraacetic acid (EDTA).

In other exemplary embodiments, where attachment of an immunological agent to a functionalized lipid is performed through conjugation of halocetamide to sulfhydryls, conjugation of the haloacetamides to the sulfhydryls can be achieved in thiol-free buffered aqueous solution at a pH of about 8.3 for about 1 to about 24 hours to form a covalent thioether linkage by nucleophilic substitution of the halogen with the thiol. According to this approach no other reagents are needed to achieve conjugation.

In other exemplary embodiments, where attachment of an immunological agent to a functionalized lipid is performed through conjugation of pyridil disulfides to sulfhydryls, conjugation of the pyridyl disulfides to the sulfhydryls can be achieved in thiol-free buffered aqueous solution over a broad pH range for about 1 to about 24 hours to form disulfide bonds. According to this approach no other reagents are needed to achieve conjugation.

In other exemplary embodiments, where attachment of an immunological agent to a functionalized lipid is performed through conjugation of thiosulfate to sulfhydryls, conjugation of thiosulfates with sulfhydryls can be achieved in thiol-free buffered aqueous solution over a broad pH range for 1 to 24 hours to form disulfide bonds. No other reagents are needed in those embodiments.

In all those exemplary embodiments, conjugation of the immunological agent with the functionalized NLP can be monitored using techniques/methods such as the ones described above.

Exemplary immunological molecule that can be presented through conjugation with any one of the anchor compounds herein described comprise peptides of SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 45), SEQ ID NO: 46, SEQ ID NO: 67, (SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73 alone or in combination one with the other and/or with protein antigens such as IglC, SucB, OmpA, and DnaK in nanolipoprotein particles further comprising at least one adjuvant and preferably at least one Francisella LPS. In particular the peptides, protein antigens and/or adjuvant can be attached to a functionalized amphipathic lipid comprising one or more of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-((folate)amino)hexanoyl), 1,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine-N-(6-azidohexanoyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine-N-(succinyl), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl), 1,2-dioleoyl-sn-glycero-3-phosphoethanol amine-N-(glutaryl), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanyl), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(hexanoylamine), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanylamine), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclhexane-carboxamide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine-N-(biotinyl), 1,2-Dioleoyl-sn-Glycero-3-Phospho(Ethylene Glycol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl, 1,2-di stearoyl-sn-glycero-3 -phosphoethanol amine-N-[dibenzocyclooctyl(polyethylene glycol)-2000], 1,2-di stearoyl-sn-glycero-3 -phosphoethanolamine-N-[succinyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3 -phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000], cholesterol modified oligonucleotides, cholesterol-PEG2000-azide, cholesterol-PEG2000-Dibenzocyclooctyl, cholesterol-PEG2000-maleimide, cholesterol-PEG2000-N-hydroxy succinimide esters, cholesterol-PEG2000-thiol, cholesterol-azide, cholesterol-Dibenzocyclooctyl, cholesterol-maleimide, cholesterol-N-hydroxysuccinimide esters, cholesterol-thiol, C18 modified oligonucleotides, C18-PEG2000-azide, C18-PEG2000-Dibenzocyclooctyl, C18-PEG2000-maleimide, C18-PEG2000-N-hydroxysuccinimide esters, C18-PEG2000-thiol, C18-azide, C18-Dibenzocyclooctyl, C18-maleimide, C18-N-hydroxysuccinimide esters, C18-thiol.

In some embodiments, attachment of one or more antigens and/or other immunological agents in a same functionalized NLP can be performed using different anchor compounds and corresponding anchor substrate for a same NLP where the selection of compatible anchor/anchor substrate pair can be performed by the skilled person in view of the immunological agent(s) to be attached, the chemistry of the compounds involved and the experimental design.

In particular, compatibility of the anchor/substrate pair of choice with all the NLP components has to be considered in selecting a suitable NLP for attaching a target molecule of interest, especially if conjugation to the lipid bilayer, rather than the scaffold protein, is performed. For example, in some embodiments amine-based conjugation is not compatible with certain scaffold proteins. If the scaffold protein in question contains a free amine group, then amine-reactive compounds will conjugate to the scaffold proteins. Similarly, compounds functionalized with reactive maleimides would react with any available sulfhydryl moieties present in cysteine-containing scaffold proteins. A skilled person will be able to identify and sort components according to a desired experimental design.

In some embodiments immunogenic NLP comprises a scaffold protein, a membrane forming lipid, a functionalized amphipathic compound, and the antigenic combination herein described. In the immunogenic NLPs, at least one protein antigen from the Francisella bacterium of the Francisella protein antigen component (either derived to comprise an anchor or attached to an anchor compound to form an anchored protein antigen) presents an anchor portion attached to the anchor compound substrate of the functionalized amphipathic compound. Antigenic polysaccharides of the antigenic Francisella polysaccharide component, such as derivatives of the antigenic LPS and/or CPS lacking the Lipid A or Lipid-A like moiety can be modified to present an anchor compound suitable for attachment to the anchor compound substrate of the functionalized amphipathic compound.

In some embodiments, the antigenic Francisella polysaccharide component and the NLP are assembled at a ratio about 1 to 50 LPS molecules per NLP, preferably 2-10 LPS molecules per NLP (see Example 5).

In some embodiments, the Francisella protein antigen component and the NLP are assembled at a ratio about 1 to 50 protein antigen molecules per NLP, preferably 2-10 protein antigen molecules per NLP (see Example 5).

In some embodiments, the adjuvant and the NLP are assembled at a ratio about 1 to 50 adjuvant molecules per NLP, preferably 2-10 adjuvant molecules per NLP.

In some embodiments, the immunogenic NLPs herein described comprise the NLPs, adjuvants, the antigenic Francisella polysaccharide component and the Francisella protein antigen component at a ratio range of 1:1:1:1: to 1:50:50:50, preferably at a ratio range of 1:2:2:2 to 1:10:10:10.

FIG. 3 shows a schematic illustration of nanoparticle incorporating a Francisella LPS and an exemplary protein antigen IglC wherein the incorporation of the IglC occurs through binding of an anchor compound with a corresponding anchor compound substrate. In particular, the illustration of FIG. 3 schematically shows the coupling of the protein antigen to the surface of a functionalized NLP bilayer. The lipid component of the LPS (lipid A) is embedded into the bilayers of the NLP acting as an anchor to conjugate the LPS molecule to the NLP, while the polysaccharide component of the LPS is presented on the surface of the NLP. As such, the LPS can be added to the NLP during the self-assembly process. NLPs assembled with a fraction of headgroup-functionalized lipids, providing a means of either covalently or noncovalently coupling the adjuvant to the surface of the NLP bilayer.

As a person skilled in the art will understand, LPS is an amphipathic molecule. In solution, LPS alone typically forms very high molecular weight aggregates. These aggregates must be dissociated into monomeric units so that individual LPS molecules (not aggregates) are available for incorporation into the NLP bilayer during self-assembly (see FIGS. 8 and 9).

In some immunogenic nanoparticles herein described, an adjuvant herein described is incorporated into the NLP. The incorporation of an adjuvant into NLPs can be achieved through binding of an anchor compound with a corresponding anchor compound substrate in a similar way a protein antigen is presented on NLPs.

Exemplary adjuvants that can be attached to an NLP herein described through anchor binding include, but are not limited to, immunostimulatory peptides (e.g. f-Met-Leu-Phe), muramyl dipeptide (including the analog muroctasin), saponins (e.g. gylcosidic terpenes from Qillaja Saponaria), toxins (e.g. tetanus, cholera), oligonucleotide CpG motifs (e.g. Agatolimod), immunostimulatory carbohydrates and polysaccharides, and immunostimulatory protein or peptide molecules (e.g. cytokines, chemokines, flagellin, and derivatives thereof).

Alternatively, an adjuvant can be synthetically appended with a long chain fatty acid, or other hydrophobic or amphipathic moieties, to form an amphipathic adjuvant having a hydrophilic portion and a hydrophobic portion. For example, a CpG molecule can be modified to include a cholesterol moiety. The cholesterol moiety anchors the highly-polar CpG molecule to the NLP. Any lipidic compound can be used with similar anchoring effect. Exemplary lipidic compound include acyl chains, hydrophobic carbon chains, and others identifiable to a person skilled in the art. Due to the amphipathic nature of this molecule, it can be added both during NLP self-assembly or after NLPs have been formed. In some preferred embodiments, the adjuvants are added after NLPs have been formed (see FIG. 3).

The immunogenic NLPs herein described can further comprise other immunological agents such as other carbohydrate antigen. For example, O-antigens of LPS can be conjugated or incorporated into the immunogenic NLPS.

In several embodiments, immunogenic NLPs herein described can be formed with a variety of phospholipids including but not limited to: dimyristoylphospatidylcholine (DMPC), dioleoylphosphoethanolamine (DOPE), dioleoylphophatidylcholine (DOPC),

dioleoylphosphoserine (DOPS), dioleoylphosphoserine (DOPS), dioleoyl-glycero-3-[(N-(5-amino-1carboxypentyl) iminodiacetic acid)succinyl] (DOGS-NTA).

In several embodiments, immunogenic NLPs herein described can be formed with apolipoproteins that include human ApoE4 22K and insect lipophorins from Bombyx mori and Manduca sexta, apoA-1, and ApoA-1 derivatives.

In several embodiments the functionalized membrane-forming lipid of the immunogenic NLP can include but is not limited to dioleoyl-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DOGS-NTA) and DOGS-NTA(Ni), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (18:1 MPB PE), and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt), and azido- and propargyl-modified lipids.

In particular, in some of those embodiments, where binding is performed by interaction with a chelated bivalent metal ion, the chelant is a modified lipid molecule, e.g. dioleoyl-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DOGS-NitriloTriaceticAcid) and DOGS-NTA(bivalent metal ion) to which His-tagged proteins can be specifically and directly conjugated.

Embodiments based on the bivalent metal ion-chelating ability of NiNLPs allow conjugation of any (His)-tagged protein, opening the door to thousands of potential immunological agents. Bivalent metal ions comprise Ni and additional transition metals bound or chelated by poly-histidine sequences. Exemplary bivalent metal ions include but are not limited to Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺. Corresponding polyhistidine tags can be comprised at either one of the ends of the target molecule to be attached. For example, a His-tag can be added at either the N- or C-terminus of recombinantly expressed proteins to enable rapid isolation and purification.

In other embodiments, the membrane-forming lipid can be functionalized to contain an azide group that can react with an immunological agent (e.g. a protein) specifically modified to contain a propargyl group. The reaction product between the azide and acetylene group forms a 1,2,3-triazole moiety. The product of this cycloaddition reaction or “click chemistry” is a covalent association between the immunological agent and the NLP.

Still in other embodiments, a thiol group is added to the immunological agent and is then reacted with a maleimide group presented on a functionalized membrane-forming lipid. Maleimide bearing lipids (functionalized anchor substrate lipid), are available commercially. In this case, an anchor-bearing immunological agent would be configured to present a free thiol group that could add to the maleimide moiety forming a covalent bond.

In particular, metal chelating lipids, such as 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (ammonium salt), and the like, that are suitable in forming NLPs herein described, are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where metal chelating lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain a polyhistidine tag for conjugation. Attachment of a polyhistidine tag to the immunological agent can be achieved through molecular biological approaches and techniques identifiable by a skilled person.

Also negatively charged headgroup lipids, such as phosphatidic acid-, phosphatidylserine, phosphatidylglycerol-bearing lipids, and the like, that are suitable in forming NLPs herein described are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where negatively charged headgroup lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain a polyarginine tag for conjugation. Attachment of a polyarginine tag to the immunological agent can be achieved through molecular biological approaches and techniques identifiable by a skilled person. Further, immunological agents that are inherently positively charged require no further modification.

Positively charged headgroup lipids, such as 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (chloride salt), and the like, that are suitable in forming NLPs herein described are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where positively charged headgroup lipids are used in a functionalized NLP, the corresponding target molecules are configured to contain an overall negative charge for conjugation sufficient to allow binding with the headgroup. In some of those embodiments the negatively charged immunological agents require no further modification to allow conjugation with the functionalized NLP.

Biotinylated lipids, such as 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (sodium salt), and the like, that are suitable in forming NLPs herein described are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where biotinylated lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain avidin (and/or derivatives thereof), via an additional biotin moiety.

Glutathione-derivatized lipids such as phosphatidylethanolamine-bearing lipid that are suitable in forming NLPs herein described be formed through coupling of glutathione to an appropriate lipid. In embodiments where glutathione-derivatized lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain a glutathione S-transferase fusion protein tag for conjugation. Attachment of a glutathione S-transferase fusion protein tag to the immunological agent can be achieved through molecular biological approaches and techniques identifiable by a skilled person.

Strep-tag II-derivatized lipids that are suitable in forming NLPs herein described can be formed through coupling of synthetic strep-tag II to an appropriate lipid, such as. phosphatidylethanolamine-bearing lipid, according to techniques identifiable by a skilled person. In embodiments where glutathione-derivatized lipids used in a functionalized NLP, the corresponding immunological agents are configured to contain a compound such as Strep-Tactin, which is commercially available.

Amine-bearing lipids, such as phosphatidylethanolamine that are suitable in forming NLPs herein described are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where amine bearing lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain an amine-reactive moiety, (e.g. active esters, isothiocyanates, sulfonyl chlorides, dichlorotriazines, aryl halides, acyl azides, and the like). Attachment of an amine reactive moiety to the immunological agent can be achieved through previously established coupling chemistries and techniques identifiable by a skilled person.

Carboxylic acid-bearing lipids, such as. 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (sodium salt), that are suitable in forming NLPs herein described are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where carboxylic acids are used in a functionalized NLP, the corresponding immunological agents are configured to contain a reactive primary amine, e.g. lysine side chain presented on the immunological agent for binding with the carboxylic acid-bearing lipids.

Thiol-reactive lipids, such as 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide] (sodium salt), that are suitable in forming NLPs herein described are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where thiol-reactive lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain a reduced sulfhydryl moiety, such as reduced cysteine residue.

Free sulfhydryl-bearing lipids, such as 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol (Sodium Salt), that are suitable in forming NLPs herein described are commercially available or synthesizable by a skilled person using techniques known in the art. In embodiments where free sulfhydryl-bearing lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain thiol-reactive moieties, such as maleimides (and derivatives), haloacetamides, pyridyldithio-propionate, and thiosulfates. Azide- and alkyne-bearing lipids that are suitable in forming NLPs herein described can be prepared from commercially available components that react with phosphatidylethanolamine-bearing lipids, e.g. 3-(azidotetra(ethyleneoxy))propionic acid, succinimidyl ester and 3-propargyloxypropanoic acid, succinimidyl ester, respectively. In embodiments where azide- and alkyne-bearing lipids are used in a functionalized NLP, the corresponding immunological agents are configured to contain either an acetylene- or azide reactive group, respectively, to form a stable 1, 2, 3 triazole reaction product.

In any of the above embodiments, one or more additional same or different adjuvant and/or antigen can be attached to the immunostimulatory nanoparticle through binding the anchor compound-anchor substrate compound and/or through self-assembly of the additional amphipathic or hydrophobic adjuvant and/or antigen in the nanoparticle assembly process and/or through binding lipidic-anchored adjuvant and/or antigen to preformed NLPs.

For example, in several embodiments, NLPs made from apolipoprotein and a mixture of DOGS-NTA (Ni) [1-50%] and DMPC, DOPC form nanoparticles containing nickel ions on the surface or face of the particle (Ni-NLPs). In some of those embodiments, protein conjugation between these Ni containing NLPs bind antigens formed by His labeled proteins (see Examples 1-3 wherein conjugation has been shown with particular reference to the major envelope protein from West Nile Virus (WNV—Env).

Accordingly, in some embodiments, the antigens and/or other immunological agents herein described including LPS and protein antigens from Francisella tularensis, and adjuvants can be presented on an NLP allowing assembly of customized immunogenic platforms comprising a combination of selected antigens and/or other immunological agents immobilized on the surface of the lipid bilayer.

A schematic exemplary representation of an immunogenic nanolipoprotein particle is illustrated in FIG. 3. In particular, FIG. 3 shows an NLP assembly with the incorporation of an adjuvant, a LPS and a protein antigen both from Francisella tularensis.

In some embodiments of the immunogenic NLPs, at least one Francisella LPS together with at least one protein antigen from Francisella are contained on an NLP with at least one adjuvant molecule. In some other embodiments, Francisella LPS and protein antigens are separately contained on NLPs with at least one adjuvant molecule. In the embodiments herein described, each NLP is conjugated with an adjuvant and preferably contains at least one Francisella LPS and/or at least one Francisella protein antigen.

Accordingly, in a population of immunogenic NLPs comprising Francisella LPS, Francisella protein antigens and adjuvants, the population of immunogenic NLPs can comprise a subpopulation of immunogenic NLPs each containing at least one protein antigen from Francisella, at least one LPS from Francisella, and at least one adjuvant molecule, and/or a subpopulation of immunogenic NLPs each containing a Francisella LPS and an adjuvant molecule, and/or a subpopulation of immunogenic NLPs each containing a Francisella protein antigen and an adjuvant molecule, and/or a subpopulation of immunogenic NLPs each containing a Francisella LPS and Francisella protein antigen.

The amount of antigens and immunological agents on the NLP biolayer surface can be controlled by the input ratios of the antigens and immunological agents, membrane-forming lipids and the functionalized membrane-forming lipids, allowing the control of immunological agent loading in the NLP.

In some embodiments, the immunogenic NLP comprises F. tularensis LVS LPS, F. tularensis protein antigen IglC, and an adjuvant CpG. The LPS is anchored to the NLP bilayer during NLP self-assembly via the hydrophobic Lipid A moiety of LPS, which anchors the LPS to the NLP. The protein antigen is conjugated to the pre-formed NLP via a polyhistidine tag on the NLP and the cognate chelated nickel present on the NLP bilayer surface. The CpG molecule is modified with a cholesterol moiety. The modified amphipathic molecule is added to pre-formed NLP, whereby the cholesterol moiety inserts into the NLP bilayer and anchors the CpG moiety to the bilayer surface. In some embodiments, the preferred ratio of NLP:LPS:protein:adjuvant is 1:2:2:2.

Immunogenic nanolipoprotein particle according to the present disclosure and NLP carrier presenting at least one of the antigenic polysaccharide from the Francisella bacterium or derivative thereof forming the antigenic Francisella polysaccharide component, the protein antigen from the Francisella bacterium or derivative thereof of the Francisella protein antigen component, the adjuvant, can be provided according to methods identifiable by a skilled person upon reading of the present disclosure.

In some embodiments a method to provide an NLP comprises: providing a membrane-forming lipid, a scaffold protein and optionally a functionalized membrane-forming lipid presenting an anchor compound substrate, together with at least one molecule of the antigenic Francisella polysaccharide component, the Francisella protein antigen component, and the adjuvant.

In these embodiments the antigenic LPS from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium, and the adjuvant comprise a hydrophobic region configured for attachment to a membrane lipid bilayer, and/or comprise an anchor compound presented for binding with the anchor compound substrate on the functionalized amphipathic compound.

In embodiments, wherein the method is directed to provide NLPs presenting at least one molecule of the antigenic Francisella polysaccharide component and the Francisella protein antigen component and the adjuvant, comprising a hydrophobic region configured for attachment to a membrane lipid bilayer, the method comprises mixing the membrane-forming lipid, the scaffold protein, and optionally the functionalized amphipathic compound, together with the antigenic LPS from the Francisella bacterium or derivative thereof, the protein antigen from the Francisella bacterium, and the adjuvant comprising a hydrophobic region.

In the method the mixing is performed for a time and under condition to allow self-assembly of the membrane-forming lipid and the scaffold protein into a discoidal membrane lipid bilayer stabilized by the scaffold protein with the at least one molecule of the antigenic Francisella polysaccharide component, the Francisella protein antigen component and the adjuvant, if any, attached to the membrane lipid bilayer through interaction of the hydrophobic region with the membrane lipid bilayer to form a nanolipoprotein particle. For example, NLPs can incorporate LPS (consisting of lipid A, core, and o-antigen), a membrane protein antigen, and a cholesterol-tagged CpG adjuvant. The LPS, membrane protein, and scaffold protein are added to surfactant-solubilized lipids. The surfactant is removed to initiate NLP self-assembly and the LPS and membrane protein are anchored to the NLP lipid bilayer by either the lipid A moiety or hydrophobic polypeptide region of the LPS or membrane protein respectively. The cholesterol-tagged CpG adjuvant can then be added to these pre-formed NLPs, whereby the cholesterol moiety will anchor the molecule to the NLP lipid bilayer.

In embodiments wherein at least one molecule of the antigenic Francisella polysaccharide component, the Francisella protein antigen component, and the adjuvant comprise an anchor compound presented for binding with the anchor compound substrate on the functionalized amphipathic compound, the method further comprises mixing a formed nanolipoprotein particle comprising a functionalized amphipathic compound presenting an anchor compound substrate with the at least one molecule of the antigenic Francisella polysaccharide component, the Francisella protein antigen component and the adjuvant for a time and under condition to allow the attaching to the nanolipoprotein particle through the binding of the anchor compound and the anchor compound substrate of the functionalized membrane-forming lipid.

In some embodiments, the method comprises providing a membrane-forming lipid, a scaffold protein, a functionalized membrane-forming lipid attached with an anchor compound substrate, and an antigenic Francisella polysaccharide component comprising a polysaccharide from Francisella or derivative thereof, the polysaccharide or derivative thereof further comprising a lipid component and a polysaccharide component; mixing the membrane-forming lipid, the scaffold protein, the functionalize membrane-forming lipid, and the antigenic Francisella polysaccharide component for a time and under condition to allow self-assembly of the membrane-forming lipid, the scaffold protein, the functionalize membrane-forming lipid into a nanolipoprotein particle with the lipid component of the Francisella polysaccharide embedded into the nanolipoprotein particle and the polysaccharide component of the Francisella polysaccharide on the surface of the nanolipoprotein particle.

The method further comprises attaching a protein antigen and/or a derivative thereof of a Francisella protein antigen component to an anchor compound thus providing an anchored Francisella protein antigen component; mixing the formed nanolipoprotein particle with the anchored Francisella protein antigen component for a time and under condition to allow the anchored a protein antigens and/or a derivative thereof of the anchored Francisella protein antigen attaching to the nanolipoprotein particle through the binding of the anchor compound and the anchor compound substrate of the functionalized membrane-forming lipid. For example, soluble protein antigens that incorporate a poly-histidine peptide tag (anchor compound) can be conjugated to a lipid-containing platform that features a lipid chelating nickel at the polar headgroup (anchor compound). The polyhistidine-tagged antigen will bind to the chelated nickel at the lipid platform surface and remain tethered.

As exemplified in the illustration of FIG. 3, purified starting components are provided, which comprise F.t. LPS, a membrane-forming lipid, a functionalized membrane-forming lipid, and a scaffold protein. The F.t. LPS comprises a hydrophilic component (polysaccharide component) and a hydrophobic component (lipid A component). The starting components are contacted for a time and under conditions to allow assembly of the LPS:NLP.

The Francisella LPS is first heated in cholate or some other surfactants prior to the addition to the NLP to ensure that monomeric LPS is present during the self-assembly reaction.

In some embodiments, the dispersed Francisella LPS, membrane-forming lipids, functionalized membrane-forming lipids, and scaffold proteins are incubated for about 30-600 minutes at a temperature ranging from 15-35° C. In some preferred embodiments, the contact time is about 60-120 minutes and the incubation temperature is from 20-25° C. (see Example 5).

The formed LPS:NLP are further mixed with a protein antigen from Francisella tularensis attaching an anchor compound and, optionally, an amphipathic adjuvant having a hydrophobic moiety and a hydrophilic moiety. The protein antigen from Francisella tularensis is attached with an anchor compound which allows the protein antigen to be attached to the NLP through the binding of the anchor compound and the anchor compound substrate from the functionalized membrane-forming lipids. The hydrophobic moiety of the adjuvant can be appended to a hydrophilic adjuvant using synthetic procedures identifiable by a skilled person or be naturally occurring. The LPS:NLP, the protein antigen from Francisella tularensis and the amphipathic adjuvant are contacted for a time and under conditions to allow assembly of the immunogenic NLP. The protein antigen and the amphipathic adjuvant are incubated for at least 5 minutes, preferably 30 minutes. The incubation can be done at temperature ranges between +2° C. and +37° C., preferably at +20° C.

As illustrated in FIG. 3, the immunogenic NLPs formed by the assembly of the membrane-forming lipids, the functionalized membrane-forming lipids, the scaffold protein, the FT LPS, the protein antigen from Francisella tularensis and optionally the adjuvant is configured to present the polysaccharide portion of the FT LPS and the protein antigen from Francisella tularensis on the surface of the nanolipoprotein particle.

Other membrane-derived vesicles such as membrane microparticles can also be used as carriers in place of NLPs for incorporating the antigens and/or adjuvants. In

some embodiments herein described, an immunogenic composition is described. The immunogenic composition comprises the antigenic combination described together with a suitable vehicle.

The term “vehicle” as used herein indicates any of various media acting usually as solvents, carriers, binders or diluents for antigens and/or NLPs comprised in the composition as an active ingredient.

The term “excipient” as used herein indicates an inactive substance used as a carrier for the active ingredients of a medication. Suitable excipients for the pharmaceutical compositions herein disclosed include any substance that enhances the ability of the body of an individual to absorb the immunogenic composition. Suitable excipients also include any substance that can be used to bulk up formulations with the composition herein described to allow for convenient and accurate dosage. In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of the immunogenic compositions. Depending on the route of administration, and form of medication, different excipients may be used. Exemplary excipients include but are not limited to antiadherents, binders, coatings disintegrants, fillers, flavors (such as sweeteners) and colors, glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which is issued to dilute or carry an active ingredient of a composition. Suitable diluent includes any substance that can decrease the viscosity of a medicinal preparation.

In some embodiments, the components of the antigenic combination are comprised in an amount effective to induce immunogenic responses in a subject. The term “effective amount” refers to a nontoxic but sufficient amount of one or more active ingredients of the antigenic combination to provide a desired effect. Accordingly, an effective amount associated with inducing immunogenic responses in a subject against bacterial infections caused by Francisella refers to non-toxic but sufficient amount of the at least one Francisella LPS and the at least one Francisella protein antigen.

The immunogenic composition can comprise an antigenic Francisella polysaccharide component (e.g. a Francisella LPS) in an amount from about 0.1 μg antigen/vaccination to about 100 μg antigen/vaccination, preferable 2-20 μg antigen/vaccination. The immunogenic composition also comprises Francisella protein antigen component in an amount from about 0.1 μg antigen/vaccination to about 100 μg antigen/vaccination, preferable 5-50 μg antigen/vaccination.

Optimal vaccine composition and immunization regimen can be determined empirically by skilled person. The general approach is to select and modify one parameter of the vaccine composition or immunization regimen while keeping all other parameters unchanged. The effect of the modification can be evaluated by immunizing separate groups of animals with the unmodified and modified vaccines and then challenging the vaccinated animals with a lethal dose of aerosolized Francisella tularensis SCHU S4 strain. The stringency of the test can be increased with larger challenge doses. Modifications that significantly improves survival, bacterial burden, or other measurable physiological parameters such as body weight, temperature, etc. compared to the unmodified vaccine will be selected for further development. Similar approaches can be used to optimize other parameters and to optimize the vaccine composition or immunization regimen for other animal species, including humans.

In particular, the immunogenic composition herein described can comprise the antigenic Francisella polysaccharide component and the Francisella protein antigen component at a ratio between 1:0.1 and 1:10 by mass. In some embodiments, the immunogenic composition comprises about 5 μg of LPS antigen and about 20 μg of protein antigen (see Examples 12-17). In some embodiments, the components of the antigenic combination and/or related subcomponents are comprised in the composition presented on one or more same or different carriers. In particular in some embodiments the antigenic Francisella polysaccharide component and the Francisella protein antigen component and/or related antigenic Francisella polysaccharides and derivatives thereof and Francisella protein antigens and derivatives thereof can be presented on single same or different carrier. In some embodiments the components and/or subcomponents of the antigenic combination can be presented on separate same or different carriers. In particular, in some embodiments, the carrier can comprise one or more membrane-derived platform. In some embodiments, the membrane derived platform carrier can be a nanolipoprotein particle.

In embodiments herein described, the immunogenic composition further comprises one or more compatible and pharmaceutically acceptable vehicles, excipients or diluents selected to provide the composition in a particular form, for example, aerosol, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, for various routes of administration.

In some embodiments, the immunogenic composition herein described are provided as pharmaceutical compositions such as a vaccine for preventing tularemia in a subject. The term “vaccine” as used herein indicates a composition, and in particular a biological preparation, that establishes or improves immunity to a particular external pathogenic assault, or an inherent transformational incident resulting in a cancerous condition in mammals. Vaccines in the sense of the present description can be prophylactic, or therapeutic.

In some embodiments herein described, the combination of the antigenic Francisella polysaccharide component and the Francisella protein antigen component can cause a synergistic effect in protection against Francisella tularensis, particularly type A Francisella tularensis. In particular, the immunogenic composition herein described can induce both a humoral immunity and a cellular immunity, thus resulting in an increase in the potency of the vaccine and enhanced protective capacity against virulent Francisella over either component alone.

In some exemplary embodiments described herein, a vaccine formulation comprising either a LPS antigen or a protein antigen from Francisella tularensis provides no protection or only marginal protection effect against Francisella tularensis. By contrast, animals vaccinated with a formulation comprising both the LPS antigen and the protein antigen from Francisella tularensis achieve significantly higher survival rate (Example 14 and 18) and less severity of symptoms such as less severe pathology (Example 13 and 18) against aerosol challenge with type A F. tularensis.

In some embodiments, when the LPS and the protein antigen from Francisella are presented in a carrier, such as a nanolipoprotein particle, to form an immunogenic nanolipoprotein particle, the immunogenic nanolipoprotein particle allows co-localized presentation of the antigens thus resulting in even more enhanced immunostimulation and immunogenic response (Examples 17 and 18). The NLP formulation appears to be superior in that animals vaccinated with NLP formulations exhibited much less severe disease as evidenced by minimal weight loss after challenge and showed much faster resolution of disease after the onset of clinical signs.

In several embodiments, the immunogenic compositions comprising antigenic combinations alone or in combination with membrane-derived platforms encapsulate key requirements for vaccine formulation: non-virulence; immunostimulation; clustered antigen presentation; simple, rapid, inexpensive production; and the means to accommodate a wide range of select-agent antigens.

In several embodiments, the immunogenic compositions herein described and related compositions, methods and systems allow cost effective and rapid development of Francisella vaccines that are safe, enable immunization with multivalent/or broad-spectrum response and at the same time, are able to elicit a high levels protection following an adequate stimulation of a host immune response.

Accordingly, in some embodiments, a method of immunizing a subject against an infection of Francisella tularensis is described. The method comprises administering an effective amount of the immunogenic compositions herein described to the subject. The term “subject” “individual” or “host” as used herein in the context of immunization includes a single biological organism wherein an immune response can occur such as animals and in particular higher animals and in particular vertebrates such as mammals and in particular human beings.

The immunogenic compositions herein described can be formulated as pharmaceutical compositions such as vaccines for administration in any suitable manner, such as intranasally, intramuscularly, intradermally, intratracheal or by other routes such as subcutaneously, intravenously, or even orally to a host. More than one route can be used to administer the immunogenic composition herein described. Some routes such as intranasal, intramuscular, or subcutaneous routes can provide a more immediate and more effective reaction than other regimens.

The dose administered to a subject should be sufficient to induce a beneficial therapeutic response in the subject over time. Amounts effective for therapeutic use depend on for example the antigen composition, the routes of administration, the weight and general state of health of the subject and other factors identifiable to a person skilled in the art. Single or multiple dosage of the immunogenic compositions can be administered simultaneously or subsequently.

In some embodiments, more than one-time immunization is required for conferring optimal protection against Francisella infections. A subsequent boost vaccination can be administrated 3-four weeks after the initial prime vaccination. Up to 6 immunizations can be administrated in a 2-year time period. Optimal vaccine composition and immunization regimen can be determined empirically by skilled person. The general approach is to select and modify one parameter of the vaccine composition or immunization regimen while keeping all other parameters unchanged. The effect of the modification can be evaluated by immunizing separate groups of animals with the unmodified and modified vaccines and then challenging the vaccinated animals with a lethal dose of aerosolized Francisella tularensis SCHU S4 strain. The stringency of the test can be increased with larger challenge doses. Modifications that significantly improves survival, bacterial burden, or other measurable physiological parameters such as body weight, temperature, etc. compared to the unmodified vaccine will be selected for further development. Similar approaches can be used to optimize other parameters and to optimize the vaccine composition or immunization regimen for other animal species, including humans.

In general, administration to an individual begins prior to the first sign of disease, or possibly at the first sign of possible exposure to F. tularensis. In some cases, administration can also be given to an individual who is early diagnosed with Francisella infection.

In certain embodiments, the immunogenic compositions and, in particular, pharmaceutical compositions, can be formulated for systemic administration, which includes parenteral administration and more particularly intravenous, intradermic, and intramuscular administration.

In some embodiments, the compositions herein described are administrated to humans or animals by intranasal inhalation. The intranasal formulations can include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary functions. Diluents such as water, aqueous saline or other substances can be employed. The nasal formulations can also contain preservatives as will be identified by those skilled in the art. A surfactant can also be included to enhance absorption of the active ingredients in the vaccine by the nasal mucosa. In some exemplary embodiments, intranasal formulations are prepared in pharmaceutical grade saline and 50 μl instilled into the nose (25 μl per nare) using a micropipette while animals were under anesthesia.

In some embodiments, the compositions herein described are administrated to humans or animals by intramuscular administration. Exemplary intramuscular formulations can be prepared in pharmaceutical grade saline and 100 μl injected into the hindleg using a TB syringe.

Exemplary compositions for parenteral administration include sterile aqueous solutions, injectable solutions or suspensions including the immunogenic composition herein described. In some embodiments, a composition for parenteral administration can be prepared at the time of use by dissolving a powdered composition, previously prepared in a freeze-dried lyophilized form, in a biologically compatible aqueous liquid (distilled water, physiological solution or other aqueous solution).

The term “lyophilization” (also known as freeze-drying or cryodesiccation) indicates a dehydration process typically used to preserve a perishable material or make the material more convenient for transport. Freeze-drying works by freezing the material and then reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas.

If a freeze-dried substance is sealed to prevent the reabsorption of moisture, the substance may be stored at room temperature without refrigeration, and be protected against spoilage for many years. Preservation is possible because the greatly reduced water content inhibits the action of microorganisms and enzymes that would normally spoil or degrade the sub stance.

Lyophilization can also causes less damage to the substance than other dehydration methods using higher temperatures. Freeze-drying does not usually cause shrinkage or toughening of the material being dried. In addition, flavors and smells generally remain unchanged, making the process popular for preserving food. However, water is not the only chemical capable of sublimation, and the loss of other volatile compounds such as acetic acid (vinegar) and alcohols can yield undesirable results.

Freeze-dried products can be rehydrated (reconstituted) much more quickly and easily because the process leaves microscopic pores. The pores are created by the ice crystals that sublimate, leaving gaps or pores in their place. This is especially important when it comes to pharmaceutical uses. Lyophilization can also be used to increase the shelf life of some pharmaceuticals for many years.

In pharmaceutical applications freeze-drying is often used to increase the shelf life of products, such as vaccines and other injectables. By removing the water from the material and sealing the material in a vial, the material can be easily stored, shipped, and later reconstituted to its original form for injection.

The antigens and/or other immunological agents of the present disclosure may be also formulated in separate dosage forms which are provided as a system that can be in the form of a kit of parts comprising any of the active ingredients and any of the formulations described herein.

Accordingly, in some embodiments, a system for immunizing a subject against an infection of Francisella is described, the system comprising at least one Francisella LPS, at least one Francisella protein antigen, and optionally an adjuvant, alone or incorporation within a membrane-derived platform for simultaneous, combine or sequential used in the methods described herein.

The system herein disclosed can be provided in the form of kits of parts. For example, the components herein described can be included as a molecule alone or in a composition together with suitable vehicle carrier or auxiliary agents.

Additional components can also be included and comprise microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure.

In the kit of parts herein disclosed, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here disclosed. In some embodiments, the kit can contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, can also be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (such as wash buffers and the like).

EXAMPLES

The methods and systems herein disclosed are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting. In particular, the immunogenic compositions comprising various LPS, protein antigens, adjuvants as well as nanolipoprotein particles were prepared at various combination and concentrations and characterized in vitro and tested in vivo. A skilled person will be able to use other lipopolysaccharides, protein antigens, adjuvants, other NLPs including different scaffold proteins and membrane-forming lipids, or other non-NLP membrane vesicles herein described.

Example 1 Exemplary LPS from a Francisella bacterium

LPS from Francisella bacteria share structural features as illustrated by the exemplary LPS herein described.

With reference to the lipid component of the Francisella LPS, the tetraacyl lipid A was shown to be present in all F. tularensis subspecies, F. novicida and F. philomiragia when bacteria were grown at 37° C. [29, 30]. The tetra-acylation of Francisella Lipid A severely limits its ability to activate innate immune cells via Toll-like receptor 4 (TLR4), in contrast to other bacterial LPS molecules.

Some Francisella lipid A exhibits temperature-regulated heterogeneity in their acyl chains such as lipid A from F. novicida lipid A. When F. novicida is grown at 18° C. (environmental temperature), lipid A acyl chains at the 2- and 2′-positions are composed primarily of C16:0 (3-OH). Conversely, when the bacterium is grown at 37° C. (mammalian host temperature), lipid A has longer acyl chains C18:0 (3-OH). Minor lipid A species with acyl chain variations include C14:0 or C18:0 fatty acyl branches instead of a C16:0 secondary chain at the 2′-position.

FIG. 5 shows exemplary Francisella LPS structure comprising lipid A and a polysaccharide core component in comparison with E. coli Kdo2-lipid A.

Different from lipid A of other gram-negative bacteria, Francisella lipid A include 1) absence of phosphate at the 4′ position as well as the modification of 1-phosphate with GalN and 2) tetraacylation of lipid A with longer acyl chains (16-18 carbons) (FIG. 5, panels A and E).

In particular, the lipid A of Francisella LPS contains a β-(1-6)-linked diglucosamine (GlcN) backbone with four long fatty acyl groups that are amide-linked with C18:0 (3-OH) at the 2- and 2′-positions and ester-linked with C18:0 (3-OH) at the 3-position (FIG. 5). The hydroxy group of the 2′-linked fatty acyl chain is further esterified with C16:0.

Unlike lipid A from other gram-negative species such as E. coli, which has two phosphates at the 1- and 4′-positions (FIG. 5, panel E), Francisella lipid A has an α-linked galactosamine (GalN) addition at the 1-position and lacks phosphate at the 4′-position (FIG. 5, panel A).

This major tetraacyl lipid A, typically observed at m/z 1665 by negative ion MALDI mass spectrometry (MS), is present in all F. tularensis subspecies, F. novicida and F. philomiragia when bacteria were grown at 37° C. The absence of negatively charged phosphate groups affects the overall charge of LPS and has implications in the organism's resistance to antimicrobial peptides.

An exemplary Lipid A of a Francisella LPS is shown in FIG. 6.

With reference to the polysaccharide component the antigenic combination can comprise LPS from LVS strain 15, OSU10, and SchuS4 strain (members of either subsp. tularensis or subsp. holarctica) with the O-antigen repeating units -2)-β-D-Qui4NFm-(1-4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1- (FIG. 5, panel C). In some embodiments the antigenic combination can comprise LPS from F. novicida strain U112 which has an antigenically distinct repeating O-antigen unit of -4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-Qui2NAc4NAc-(1).

Reflecting the structural variation, the O-antigen gene clusters of F. tularensis and F. novicida differ in a number of genes involved in the biosynthesis of individual O-antigen sugars. The O-antigen gene cluster of F. tularensis contains 19 putative O-antigen genes. The O-antigen gene cluster of F. novicida contains 14 putative O-antigen genes. Functional differences demonstrate that Ft LPS is important for maintaining an intracellular niche whereas F. novicida LPS appears to be important for serum stability [31, 32].

Several Francisella bacteria also comprise lipid A in free form and not attached to Kdo, core oligosaccharide, or O-antigen. The sugar and fatty acid composition of this free lipid A is very similar to lipid A with O-antigen. Both F. tularensis and F. novicida are reported to have free lipid A. In F. novicida, a small amount of free lipid A contains an α-linked glucose at the 6′-position of distal glucosamine However, the presence of O-antigen is expected to be the key determinant of a protective antigen. While some lipid A in free form may be present in the preparations of LPS isolated from Francisella tularensis, this proportion relative to molecules consisting of lipid A, core-oligosaccharide, and O-antigen is low.

Example 2 Biosynthesis of Francisella LPS

FIG. 7 illustrates a cartoon representation of Francisella LPS and its modifying proteins. For example, LpxD1 and LpxD2 are identified as N-acyltransferases whose expression and activity increases at 37° C. and 18° C., respectively. LpxL is identified as a Kdo-dependent acyltransferase responsible for modifying the secondary chain on the 2′-acyl group (FIG. 4). This function has been confirmed by demonstration of the ability of Francisella LpxL to complement the acylation of lipid A in an E. coli lpxL mutant.

A Kdo hydrolase system in Francisella is composed of kdhA and kdhB gene products. [33] and [34] In particular, in Francisella the Kdo hydrolase system is involved in removing the second side chain of two Kdo sugars initially synthesized with the Francisella LPS. More particularly, KdhA and KdhB are inner-membrane proteins and are both required for Kdo hydrolase activity in Francisella. KdhA catalyzes the cleavage of the side-chain Kdo and has homology to sialidase-family enzymes KdhB's function is unknown, but it is predicted to contain a number of alpha-helical transmembrane domains. A similar two-component Kdo hydrolase system has been identified in two other pathogens, Helicobacter pylori and Legionella pneumophila. [33] [35] [1]

Example 3 Biology of Francisella LPS

The lack of immune recognition of Francisella lipid A has been attributed to several structural differences compared to E. coli lipid A, a potent immune activator. These include 1) absence of phosphate at the 4′ position as well as the modification of 1-phosphate with GalN and 2) tetraacylation of lipid A with longer acyl chains (16-18 carbons); E.coli's lipid A is hexaacylated with shorter acyl chains (12-14 carbons) (FIGS. 5A & E). The lack of phosphate affects the overall charge of lipid A, a frequent target of cationic antimicrobial peptides. Supporting this notion that charge is important to the biology of lipid A, lpxD1,flmK, and naxD mutants of F. novicida, which are unable to modify 1- or 4′-phosphate, show increased sensitivity to polymyxin B and attenuation in mice.

Dynamic regulation of lipid A acyl chain length by LpxD1/LpxD2 at varying temperatures appears to be important in maintaining membrane permeability and susceptibility to cationic antimicrobial peptides as well. Consistent with LpxD1's optimum activity at 37° C., the lpxD1 mutant is unable to modify lipid A with longer acyl groups and is severely attenuated in mice, whereas the lpxD2 mutation has no effect.

Example 4 Preparation of Francisella LPS

LPS can be prepared by extraction from TCA, phenol, or phenol-chloroform-petroleum ether (for rough strains). TCA extracted lipopolysaccharides are structurally similar to the phenol extracted ones, with similar electrophoretic patterns and endotoxicity. The primary differences are in the amounts of nucleic acid and protein contaminants remaining after extraction. The TCA extracts contain ˜2% RNA and ˜10% denatured proteins, while phenol extracts contain up to 60% RNA and <1% protein. Subsequent purification by gel filtration chromatography removes much of protein present in the phenolextracted LPS, but results in a preparation that contains 10-20% nucleic acids. Further purification using ion exchange chromatography yields a lipopolysaccharide product which contains <1% protein and <1% RNA.

In an exemplary embodiment, Francisella LPS can be purified from F. tularensis LVS as follows. Briefly, a 1.5-liter culture of Ft LVS is grown in 4-liter flasks at 37° C. for 48 hours in Trypticase soy broth supplemented with cysteine. Following centrifugation and PBS, methanol and acetone washes, the samples are lyophilized. LPS is then extracted by the hot phenol method followed by enzymatic treatment to remove contaminants. Pellets are harvested by centrifugation for 12 hours [36].

Example 5 Assembly tests with F. tularensis live vaccine strain (LVS) LPS

Preliminary experiments were firstly conducted to incorporate LPS into NLPs using F. tularensis LVS LPS as a surrogate to evaluate if NLPs are formed and if HPLC can be used to detect the LPS in the NLP samples.

A number of assembly tests on F. tularensis LPS were then conducted following these preliminary experiments. The F. tularensis LPS were isolated from the LVS strain.

In the first set of assembly test, LPS rehydrated in water was added to cholate-solubilized lipids (30 mM cholate). Three different LPS:NLP ratios were tested: no LPS (0 μg LPS/μg rE4), a low amount of LPS (0.29 μg LPS/μg rE4), and a high amount of LPS (0.86 LPS/μg rE4).

In the second set of assembly test, LPS rehydrated in water was added to lipids solubilized in cholate (30 mM or 60 mM) or Z3-14 detergent (30 mM). Three different LPS:NLP rations were tested: no LPS (0 μg LPS/μg rE4), a low amount of LPS (0.29 μg LPS/μg rE4), and a high amount of LPS (0.86 μg LPS/μg rE4).

For each assembly test, scaffold protein was then added to the solubilized LPS-lipid and allowed to incubate for 1 hour at room temperature. After thorough dialysis to remove residual cholate, the assemblies were analyzed by size exclusion chromatography.

The results show opalescent solutions, which is an indication that there are significantly large lipid particles in solution, much large than the NLPs. This suggests that the assembly reaction did not result in NLP formation.

A third assembly test was then conducted in which detergents (cholate at 60 mM or Z3-14 at 60 mM) were first added to LPS stock (LPS rehydrated in water) and incubated for about 2 minutes at elevated temperatures prior to mixing with NLP assembly reaction components. Three incubation temperatures were tested: 37° C., 55° C. and 95° C. After thorough dialysis to remove residual cholate, the assembly was analyzed by size exclusion chromatography. The results show that incubating LPS in detergent at 95° C. results in NLP formation (FIG. 8).

A fourth assembly test was then conducted in which cholate at 60 mM was added to LPS stock and incubated at 95° C. for different incubation times: 2 minutes, 5 minutes and 15 minutes. The LPS stock mixed with cholate was then added to lipid, and then mixed with scaffold protein. After thorough dialysis to remove residual cholate, the assembly was analyzed by size exclusion chromatography. The results show that incubating LPS in cholate at 95° C. for 15 minutes was optimal for NLP formation (FIG. 9).

A fifth assembly test was then conducted in which cholate at 60 mM was added to LPS stock and incubated at 95° C. for 15 minutes. The LPS stock mixed with cholate was then added to dry lipid. Three different LPS:NLP ratios were tested: no LPS (0 μg LPS/μg rE4, NP1080), a low amount of LPS (0.29 μg LPS/μg rE4, NP1079), and a high amount of LPS (0.86 μg LPS/rE4, NP1078). After thorough dialysis to remove residual cholate, the assembly was analyzed by size exclusion chromatography. The results show successfully NLP formation in all three cases (FIG. 10). In particular, a higher amount of LPS results in larger NLP in size.

Example 6 RP-HPLC Quantification of LVS-LPS

In this example, a method was developed to characterize the incorporation of LPS into the NLP in order to quantify and control the amount of LPS incorporated in the final vaccine formulation.

This method is based on reversed-phase high-performance liquid chromatography (RP-HPLP). Using a C8 RP column, methods were developed using an aqueous buffer and two organic buffers (acetonitrile and isopropanol) to successfully separate LPS from other NLP constituents. An evaporative light scattering detector was used to detect the LPS. Standards of pure F.t. LPS were used to prepare a standard curve for quantification of unknown samples.

In particular, FIG. 11A shows the chromatograms of LPS standards at different concentrations. Peak areas were calculated for each standard. FIG. 11B shows the standard curve correlating LPS peak area with concentration of LPS. This standard curve was used to quantify LPS amounts in unknown samples.

NLPs prepared with or without increasing amount of LPS were purified by SEC. Fractions corresponding to the NLP peak were pooled and analyzed by RP-HPLC. The presence of LPS in the NLP peak fractions was verified, and subsequently quantified (FIGS. 11A-B).

Successful LVS LPS incorporation was detected as shown in Table 5.

TABLE 5 Exemplary LVS:NLP quantification μg in μg in Formed NLPs rxn NLP Yield NP1078 422 101 24% NP1079 84 36 43% NP1080 0 0 n/a

For example, in NP1078, 422 μg LVS LPS were added to the self-assembly reaction mixture and 101 μg was measured in the resultant NLPs purified by SEC. This represents a 24% yield under these conditions. In NP1079, 84 μg LVS LPS were added to the self-assembly reaction mixture and 36 μg was measured in the resultant NLPs purified by SEC. This represents a 43% yield under these conditions.

Example 7 The Binding of LPS-Bearing NiNLPs to IOC

To demonstrate that NLPs containing LPS can be used for vaccination tests in conjunction with protein antigens, NLPs were prepared with nickel-chelating lipid to form NiNLPs. The NiNLPs are able to conjugate any his-tagged protein antigens for the vaccination tests. The LPS-bearing NiNLPs with LPS at different amount were prepared according to Table 4.

It is demonstrated by using size exclusion chromatography to look at the retention of individual components and the complexes formed by adding the IglC to the NiNLP (+/−LPS) (FIG. 12A) that NiNLPs with increasing amounts of LPS are able to immobilize the His-tagged F.t. protein antigen IglC. When IglC is added to the NiNLPs, an increase in the NLP peak intensity was observed. No free IglC was observed. A shift to decreased retention times was also observed, indicating larger NLP species due to protein binding. For the NLPs with high amounts of LPS, the shift to larger size is less apparent, due to the decreased resolution of the SEC column at this size regime. These data demonstrate that the presence of LPS in the NLP does not interfere with the incorporation or conjugation of additional biomolecules.

Example 8 Concentration of LPS-NLPs

LPS-bearing NLPs are different in physical and chemical composition than traditional non-LPS-bearing NLPs. The large polysaccharide chains extending from the surface of the NLP into solution can conceivably impact the ability to concentrate the NLP solutions.

In this example, the recovery of apolipoprotein E and LPS after concentrating NLPs with increasing amounts of LPS was assessed. 200 μl of dilute NLP (pooled) was used to test effects of concentration. Vivaspin concentrators were used for concentration of samples. Scaffold protein and LPS were quantified by nanodrop and RP-HPLC, respectively. The results are shown in Table 6.

TABLE 6 ApoE and LPS recovery percentage in exemplary LPS-bearing NLPs LPS-bearing Pre-con, Post-con, LPS:apoE ratio NLPs μg μg % recovery (mg/mg) apoE NP1080 35.9 35.8 100% 0.00 NP1079 41.0 39.5 96% 0.21 NP1078 41.5 37.9 91% 0.38 LPS NP1080 0.0 0.0 n/a NP1079 9.6 8.4 88% NP1078 27.0 14.6 54%

The results show that NLPs can be successfully concentrated with an acceptable loss in material. In particular, ApoE recovery is above 90% in all cases. LPS recovery decreased as more LPS was present in the NLP. This could indicate that some of the LPS molecules may be pulled out of the NLP during the concentration process.

Example 9 Scale-Up Assembly of LVS LPS:NLP

In this example, the assembly process of forming the LPS-NiNLP was scaled up to verify that this process could be scaled for subsequent use in animal experiments.

The following parameters were used in the scale-up assembly: rE4.22k 65:35 DOPC:DOGS-NTA-Ni, 5 mg assembled with 2.7 mg LVS LPS, 15min boil in 60 mM cholate then added to dry lipid. The mixture is then purified on semi-prep Superose6 SEC column.

In general, the results show that the scale of the assemblies was increased ˜10-fold. The large assembly was split into two SEC purification runs, which appear identical (FIG. 13A). Concentrating pooled NLP fractions did not impact the shape of the SEC chromatogram (FIG. 13B). Yields were assessed as shown in Table 7.

TABLE 7 Yield in scale-up assembly of LVS LPS:NLP NLP assembly NLP product Yield rE4   5 mg  2.4 mg 48% LVS LPS 2.7 mg 0.37 mg 14% LPS:rE4 Ratio (w/w) 0.54 0.15 n/a

Example 10 Lyophilization Test of LPS:NiNLPs

Identifying appropriate storage conditions is paramount for ensuring material is available for animal experiments. In this example, two samples, lyophilized NLPs ample and non-lyophilized NLPs sample were prepared as follows. For the lyophilized NLPs sample, 4.54, NLP was formulated with 1M trehalose (0.5 μL) to reach a final concentration of 0.1M trehalose. The sample is then frozen on dry ice and lyophilized. The lyophilized LPS:NiNLPs were rehydrated in 5 μL and assessed on an analytical Superose 6 SEC column. For the non-lyophilized NLPs sample, 4.5 μL NLP was formulated with 0.5 uL H₂O, stored at 4° C., and assessed on INaSUP6 SEC column. The SEC results in FIG. 14 show no difference between the lyophilized NLPs sample and the non-lyophilized NLPs sample, suggesting that LPS:NiNLPS can be successfully lyophilized.

Example 11 Verification of Final NLP Vaccine Formulations

The final vaccine formulations to be tested are comprised of NLPs containing LPS, IglC, and the adjuvant CpG. It was previously demonstrated that His-tagged IglC could readily conjugate to LPS:NiNLPs. In this example, it is demonstrated that cholesterol-tagged CpG could also be included in the NLP formulation. In particular, IglC and CpG can be conjugated to control NLPs (NiNLPs, no LPS) (FIG. 15A) and LPS:NiNLPs (FIG. 15B).

FIG. 15A shows the his-tagged protein antigen (IglC) and cholesterol-tagged CpG can be successfully conjugated to NiNLPs. This is the control NiNLP sample that does not contain LPS, and demonstrates the expected trend in the chromatogram upon conjugation of his-tagged protein and CpG adjuvant.

FIG. 15B shows the his-tagged protein antigen (IglC) and cholesterol-tagged CpG can be successfully conjugated to LPS:NiNLPs. The same trend in the chromatogram (e.g. increase in absorbance intensity and shift to earlier elution times) is observed in NiNLP in the presence or absence of incorporated LPS. This verifies that the LPS incorporated into the NiNLP does not negatively impact the incorporation of other biomolecules.

In each case, the IglC and CpG readily conjugate to the NLP. Accordingly, desired vaccine formulation that contains NLPs functionalized with LPS, IglC, and CpG can be prepared.

Example 12 SDS-PAGE and Western Blot Analysis of LPS and LPS:NiNLPs

To further verify that LPS was incorporated into the NLP, an LPS standard and LPS:NLPs were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and probed with an antibody that specifically binds to F.t. LPS (Western Blot). The LPS:NLPs were first purified by SEC, and the fractions corresponding to the NLP peak were pooled prior to loading on the SDS-PAGE gel.

Both LPs and LPS:NLPs exhibited the expected banding pattern, indicating that a LPS molecules spanning a significant size range were present in both samples (FIG. 16). The results confirm that LPS:NLPs contain LPS and encompass the full size range of the LPS species present in the purified LPS samples used for the assembly.

A more in-depth study was also conducted to identify the types of LPS molecules that were incorporated in NLPs of different sizes. The LPS:NLPs were run on an SEC column to separate out the NLP species by size (larger species eluting first, smaller NLP species eluting later). Each of the NLP fractions was again run on analytical SEC to demonstrate the relative size of the NLPs in each purified fraction (FIG. 17B). As expected, NLPs in the earlier fractions are larger (earlier elution) than the later fractions. These fractions were also analyzed by SDS-PAGE and western blot (FIG. 17C). Based on the western blot analysis, the larger NLPs were enriched in the larger LPS species (primarily higher MW bands were observed in those fractions). In the smaller NLPs, only shorter LPS species were observed. Also, the NLP scaffold protein (ApoE) was observed in the NLP species, verifying the present of all NLP components in the purified fractions. This observation suggests a way of separating large LPS species from small LPS species.

Example 13 NLP Vaccine Formulation and Design of In Vivo Preliminary Experiments

Various experiments were designed to identify if the full NLP vaccine formulation that incorporates the protein antigen (IglC), the polysaccharide antigen (LPS) and the adjuvant (cholesterol-modified CpG) can protect the animal model (Fischer 344 rats) against a lethal challenge of virulent F.t. Adjuvanted NLPs that contained only the IglC antigen or only the LPS antigen were also tested. Two separate routes of vaccination were tested for the full NLP formulation: intranasal (i.n.) and intramuscular (i.m.). The details about the experimental design are listed in Table 8.

TABLE 8 Experimental design of exemplary vaccine formulations Animal Vaccination Anti- numbers Vaccination Vaccination Formulation Vaccination volume LPS (F344 Group # Group ID Antigen Adjuvant Platform Route (μL) mAb rat) 1 Naïve PBS I.N./I.N. 50 6 2 CpG:LPS:NLP LPS CpG NLP I.N./I.N. 50 8 (5 μg) ODN 1826 (5 μg) 3 CpG:IglC:NLP IglC CpG NLP I.N./I.N. 50 8 (20 μg) ODN 1826 (5 μg) 4 CpG:IglC:LPS: LPS CpG NLP I.N./I.N. 50 8 NLP  (5 μg); ODN IglC 1826 (20 μg) (5 μg) 5 CpG:IglC:LPS: LPS CpG NLP I.M./I.M. 100 8 NLP  (5 μg); ODN IglC 1826 (20 μg) (5 μg)

At day 0, the 344 rats in groups 1-5 were administrated with the formulations listed in Table 8. At day 28, a second administration was performed with the same formulation. At day 35, the serum of the 344 rats were collected for analysis. All rats were challenged with either intranasally or intramuscularly on day 56 with 130±65 colony forming units (CFUs) F.t. aerosol as assessed by lung deposition from three animals exposed at the same time. Survival was monitored for 21 days after challenge, at which point the lungs and spleens of the rats were collected for further analysis and assessment.

Female Fischer F344 rats (8 animals/group) were allotted into five vaccination groups: 1) PBS control, intranasal vaccination (IN); 2) CpG:LPS:NLP, IN; 3) CpG:IglC:NLP, IN; 4) CpG:IglC:LPS:NLP, IN; 5) CpG:IglC:LPS:NLP, intramuscular vaccination (IM). Animals were vaccinated with an initial prime on day 0 followed by an additional vaccination, or boost, 4 weeks later (day 28). Serum was collected from vaccinated animals one week after the boost by tail vein bleeds. Animals were challenged with 130±65 CFUs of aerosolized F. tularensis SCHU S4 four weeks after the boost (day 56). Animals were monitored for signs of clinical disease over the course of 21 days at which point the study was terminated (day 77). Surviving animals were euthanized and bacterial burdens were quantified in lungs and spleen.

Example 14 Test of Animal Survival Rate Over Time

Animals were monitored daily after challenge for survival and weights. As shown in FIG. 18, only animals vaccinated with LPS:IglC:CpG:NLPs were protected, with intranasal vaccination achieving 100% survival. Single antigen formulations using only the IglC antigen or only the LPS antigen were not protected. The data suggests that vaccine formulations were protected only when both LPS and IglC antigens were included

Example 15 Test of Animal Weight Loss Over Time

In additional to survival rate, individual animal weights were measured over the course of the experiment. These are plotted in FIGS. 19A-B for each experimental group. Each trace corresponds to the weights of an individual animal over time. In animals that did not survive (e.g. groups 1, 2, and 3) (FIG. 19A), weights dropped significantly before animals died. For the two survival groups with complete or significant survival (groups 4 and 5) (FIG. 19B), weights also dropped, but not as dramatically as in groups 1-3. In general, the weight loss over time for the intranasal (i.n.) groups was less severe than for the intramuscular (i.m.) group. The data in this example indicates that not only is survival outcome greater for the animals vaccinated with the full formulation via the i.n. route, but symptoms (i.e. weight loss) were also substantially ameliorated.

Example 16 Bacterial Tissue Burdens in Surviving Animal

After the conclusion of the 21-day survival window of the in vivo experiment, animals were culled and the lungs and spleens of the survivors were collected. These organs were assessed for the presence of bacteria by plating organ homogenates on agar plates, and resulting bacterial colonies were counted.

As shown in FIG. 20, low bacterial burdens were measured in lungs and spleens 21-days post challenge in both survival groups (groups 4 and 5). The low numbers of bacteria suggest that the animals were clearing the infection and historical data suggests that the bacterial burden would have continued to decline over time. Bacteria assessment was not performed on the animals in Groups 1-3, but typically animals that died from tularemia usually have 10⁹ or 10¹⁰ bacteria present in the lungs and spleen.

Example 17 NLP Vaccine Formulation and Design of In Vivo Confirmatory Experiments

The goal of this confirmatory experiment was to repeat the in vivo preliminary study in its entirety. In addition to the five groups tested, a non-NLP control group (group 6) was included. This group included the LPS, IglC, and CpG, but in the absence of the NLP. This group was included to demonstrate that these vaccine formulations are superior when incorporated on an NLP platform. In particular, the vaccination formulation of Group 6 comprises 20 μg IglC, 5 μg LVS LPS and 5 μg cCpG1826 and was administrated by i.n.

At day 0, the 344 rats in groups 1-6 were administrated with the formulations listed in Table 8 and described above. At day 28, a second administration was performed with the same formulation. At day 35, the serum of the 344 rats were collected for analysis. All rats were challenged with either intranasally or intramuscularly on day 56 with 130±65 CFUs F.t. aerosol. Survival was monitored for 21 days after challenge, at which point the lungs and spleens of the rats were collected for further analysis and assessment.

Female Fischer F344 rats (8 animals/group) were allotted into six vaccination groups: 1) PBS control, intranasal vaccination (IN); 2) CpG:LPS:NLP, IN; 3) CpG:IglC:NLP, IN; 4) CpG:IglC:LPS:NLP, IN; 5) CpG:IglC:LPS:NLP, intramuscular vaccination (IM); 6) CpG+IglC+LPS, IN. Animals were vaccinated with an initial prime on day 0 followed by an additional vaccination, or boost, 4 weeks later (day 28). Serum was collected from vaccinated animals one week after the boost by tail vein bleeds (day 35). Animals were challenged with 130±65 CFUs of aerosolized F. tularensis SCHU S4 four weeks after the boost (day 56). Animals were monitored for signs of clinical disease over the course of 21 days at which point the study was terminated (day 77). Surviving animals were euthanized and bacterial burdens were quantified in lungs and spleen.

The results from the quality control assessment of the vaccine formulations were shown in FIGS. 21A-B. The formulations were assessed by SDS-PAGE and probed with either a protein stain (FIG. 21A) or an antibody against F.t. LPS (Western blot, FIG. 21B). Each formulation contained the appropriate constituents, depending on the formulation. For example, the NLP formulations 2, 3, 4 and 5 all exhibited the scaffold protein by SDS-PAGE (FIG. 21A). Formulations 3, 4, 5 and 6 contained the IglC protein antigen, as verified by SDS-PAGE (FIG. 21A). Formulations 2, 4, 5 and 6 were confirmed to contain LPS by Western blot (FIG. 21B). Therefore, it is verified that the components in the formulations were present in the vaccine material used to inject the animals.

Example 18 Animal Test Results from In Vivo Confirmatory Experiments

As shown in FIG. 22, the results from animal survival test confirm the preliminary results, indicating that only animals vaccinated with formulations containing LPS, IglC and cpG were protective. Intranasal (i.n.) vaccination achieved 100% survival, but only when conjugated to NLP. Non-NLP formulations administered i.n. were ˜85% protective. Intramuscular (i.m.) administration of the complete NLP formulation provided ˜65% protection. Single antigen formulations using only the IglC antigen or only the LPS antigen were not protective or only marginally protective.

The results from these confirmatory experiments further indicate that vaccine formulations were protected only when both LPS and IglC antigens were included. NLP formulations were more protective than non-NLP formulations administered by i.n.

As shown in FIG. 23, the results from the animal weight loss test between the preliminary experiments and the confirmatory experiments follow nearly identical trends. The comparison between group 4 and group 6 also suggests that animals vaccinated i.n. with the full NLP formulation overall had less severe weight loss than those receiving the non-NLP formulation i.n.

FIGS. 24A-B provide a graphical representation of the daily clinical scores recorded during the 21-day observation period after the F.t. challenge.

Individual animals assessed based on set metrics, and scored from 0 to 5 depending on severity of symptoms. A score of 0 means no clinical signs of infection were observed (green), whereas a score of 5 means the animal has succumbed to infection (red). As expected, the animals in groups 1-3 all died (except for animal #4 in group 2) (FIG. 24A), mirroring the survival plot. In group 4 (the full NLP formulation delivered by i.n.) (FIG. 24B), only 4 animals have scores higher than 0, and only for 1 observation. In contrast, the animals in group 6 (receiving all component delivered i.n., with no NLP) (FIG. 24B) had significantly more severe clinical scores, indicating that while only 1 animal succumbed to the infection, the majority has more severe manifestation of the disease. This underscores the enhanced protective effects of the formulations prepared with the NLP.

Example 19 Extraction and Purification of CPS of F. Tularensis

Bacteria are grown for approximately 48 h on chocolate agar medium plates at 37° C., collected and suspended in a solution of 6 mM Trizma base, 10 mM EDTA, and 2.0% SDS (w/v), pH 6.8 supplemented with 50 mg/ml proteinase K, and incubated for 1 hour at 65° C. and then overnight at 37° C. Precipitate CPS with one tenth the volume of 0.3 M sodium acetate (0.1 ml per 1 ml sample) followed by 3 volumes cold 100% ethanol, flash-cooling in a dry ice-ethanol bath, and an overnight incubation at −20° C. Pellet CPS by centrifugation (10 min at 12,000×g at 4° C.). Resuspend pellets in deionized water. Repeat precipitated two additional times. Suspend samples in 15 mL buffer (10 mM Tris pH 7.4 containing 10 mM CaCl2) and treat with 80 U micrococcal nuclease overnight at 37° C. Add an equal volume of 95% phenol, incubate at 65° C. for 30 min, the cool on ice. Centrifuge the sample at 2,000×g for 10 min at 4° C. Collect the aqueous layer, then remove residual phenol by adding one-tenth volume of 0.3 M sodium acetate, followed by precipitation three times with 3 volumes of absolute ethanol. Resuspend the precipitated sample with distilled water and Triton X-114 (final concentration of 5% (v/v)), vortex, and incubated overnight at 4° C. Equilibrate at 37° C. for two hours, then centrifuge at 2000×g at 37° C. for ten minutes. Remove the aqueous upper layer, dialyze extensively against distilled water at room temperature and lyophilize. For final purification, lyophilized capsule can be reconstituted in 6 mM Tris base, 10 mM EDTA and 2.0% SDS (w/v), pH 6.8 injected onto and placed over a 1×20 cm Sephacryl SH500 column at 37° C. Purified CPS material can be characterized using a variety of techniques including gel electrophoresis, ProQ Emerald glycoprotein stain or monoclonal antibodies (e.g. mAb 11B7) for Western blotting, MALDI Mass Spectrometry (MS), gas-chromatography coupled with MS, and anion exchange chromatography as will be understood by a skilled person.

Example 20 BLAST Analysis of Exemplary Francisella Protein Antigens

A BLAST analysis of exemplary Francisella protein antigen listed in Table 2 was performed. FIG. 26 shows the search parameters and databases used for querying the protein sequences. In particular, the search was limited to Francisellacease and excluded sequences derived from unculturable/environmental samples.

The results are illustrated in FIGS. 27A to 43B. For each query sequence, a grayscale representation of a graphic summary of the BLASTP search results and representation of an alignment hit table generated from the BLASTP search, are provided. The alignment hit table shows the summary information for each target sequence found by the alignment search. Each row in the table represents a hit. For each hit sequence, the following information is included: Query ID, accession number of the hit sequence, percentage identity, alignment length, number of mismatched residues, gap open penalty, the start position in the query sequence, the end position in the query sequence, the start position in the hit sequence, the end position in the hit sequence, E-value, bit score, and percentage similarity (positivity).

Example 21 Peptide Design Rationale

For the initial screen, peptides were designed to be 15 amino acids in length and sequentially shifted in sequence by 5 residues. 15 amino acids is the approximate length estimated to be required to contain a CD4 or a CD8 epitope regardless where they start in the sequence.

The protein antigen IglC (SEQ ID NO: 2) was then divided according to the above criteria into 51 IgC protein overlapping peptides. These division can be computed with software such as Microsoft Excel using the MID function, which provides the specific number of characters from a text string (starting position and number of characters can be specified).

The resulting peptides are listed in Table 9 below.

TABLE 9 sequences of peptides 1-51 mapped from an exemplary protein antigen Ig1C Peptide No. Peptide sequence SEQ ID NO  1 MIMSEMITRQQVTSG 30  2 EMITRQQVTSGETIH 31  3 RQQVTSGETIHVRTD 32  4 TSGETIHVRTDPTAC 33  5 TIHVRTDPTACIGSH 34  6 RTDPTACIGSHPNCR 35  7 TACIGSHPNCRLFID 36  8 GSHPNCRLFIDSLTI 37  9 NCRLFIDSLTIAGEK 38 10 FIDSLTIAGEKLDKN 39 11 LTIAGEKLDKNIVAI 40 12 GEKLDKNIVAIDGGE 41 13 DKNIVAIDGGEDVTK 42 14 VAIDGGEDVTKADSA 43 15 GGEDVTKADSATAAA 44 16 VTKADSATAAASVIR 45 17 DSATAAASVIRLSIT 46 18 AAASVIRLSITPGSI 47 19 VIRLSITPGSINPTI 48 20 SITPGSINPTISITL 49 21 GSINPTISITLGVLI 50 22 PTISITLGVLIKSNV 51 23 ITLGVLIKSNVRTKI 52 24 VLIKSNVRTKIEEKV 53 25 SNVRTKIEEKVSSIL 54 26 TKIEEKVSSILQASA 55 27 EKVSSILQASATDMK 56 28 SILQASATDMKIKLG 57 29 ASATDMKIKLGNSNK 58 30 DMKIKLGNSNKKQEY 59 31 KLGNSNKKQEYKTDE 60 32 SNKKQEYKTDEAWGI 61 33 QEYKTDEAWGIMIDL 62 34 TDEAWGIMIDLSNLE 63 35 WGIMIDLSNLELYPI 64 36 IDLSNLELYPISAKA 65 37 NLELYPISAKAFSIS 66 38 YPISAKAFSISIEPT 67 39 AKAFSISIEPTELMG 68 40 SISIEPTELMGVSKD 69 41 EPTELMGVSKDGMRY 70 42 LMGVSKDGMRYHIIS 71 43 SKDGMRYHIISIDGL 72 44 MRYHIISIDGLTTSQ 73 45 IISIDGLTTSQGSLP 74 46 DGLTTSQGSLPVCCA 75 47 TSQGSLPVCCAASTD 76 48 SLPVCCAASTDKGVA 77 49 CCAASTDKGVAKIGY 78 50 STDKGVAKIGYIAAA 79 51 GVAKIGYIAAA 80

A peptide library with the peptides of Table 9 was prepared by a commercial provider.

Example 22 Identification of IglC-Specific Immune Epitopes in Fischer 344 Rats

Elucidating derivatives that may be protective in an efficacy evaluation can include mapping immune epitopes for the F. tularensis antigen. This example describes an approach to identify immune epitopes for F. tularensis IglC in the Fischer 344 rat model. The outlined approach was conducted using a live-attenuated bacterial delivery platform that overexpresses IglC (Lm-IglC) but represents an example of a broader approach to identify epitopes and derivatives of protective F. tularensis antigens.

Rats were immunized IM twice with 1×10⁸ cfu of a live-attenuated bacterial delivery platform that overexpresses IglC (Lm-IglC) followed by one IV vaccination with 1×10⁸ cfu Lm-IglC. Vaccinations were administered four weeks apart, and immune responses measured in spleens seven days after the last vaccination. Splenocytes were stimulated by individual 15aa peptides from an overlapping peptide library to IglC (MS56) and immune responses measured by IFN-γ ELISpot assay (FIG. 44). Several peptides (peptides #4, 8, 9, 16, 17, 38, 39, 43, and 44) in five different regions of the protein emerged as potentially containing IglC-specific epitopes (FIG. 44).

To determine if these responses were due to either CD4+ or CD8+ epitopes, one Fischer 344 rat was vaccinated with 1×10⁸ cfu Lm-IglC IV and boosted after two weeks with 1×10⁷ cfu Lm-IglC IV. Six days after the boost vaccination, immune responses were measured in isolated splenocytes by intracellular cytokine straining (ICS) after stimulation with peptides from each region, #4, 9, 16, 38, and 44 or no peptide as a control (F-5) (FIG. 45). Peptide #9 was identified as stimulating a CD8-mediated response.

Fine-mapping of the peptide #9-specific response was performed using 9mer peptides that overlap the entire IglC peptide #9 (FIG. 46).

In particular, for the fine mapping of peptide #9, peptides were designed to be 9 amino acids in length, sequentially shifted in sequence by 1 residue. This allowed for the identification of the exact 9 AA peptide corresponding to the T-cell epitope.

The peptides listed in Table 10 were identified and synthesized.

TABLE 10 Sequences further mapped from peptide #9 in Table 10 Peptide No. Peptide Sequence SEQ ID NO 9 NCRLFIDSLTIAGEK 38 9.1 NCRLFIDSL 81 9.2 CRLFIDSLT 82 9.3 RLFIDSLTI 83 9.4 LFIDSLTIA 84 9.5 FIDSLTIAG 85 9.6 IDSLTIAGE 86 9.7 DSLTIAGEK 87

One Fischer 344 rat was vaccinated with 1×10⁸cfu Lm-IglC IV and boosted after six weeks with 1×10⁷ cfu Lm-IglC IV. Six days after the boost vaccination, immune responses were measured in the spleen by ICS. Peptide 9.1 stimulated an immune response as great as the full-length peptide #9, while peptides 9.2 through 9.7 did not stimulate a response. Based on these data, peptide 9.1 (NCRLFIDSL (SEQ ID NO:81) was identified as an IglC-specific CD8+ T cell epitope in Fischer 344 rats (FIG. 46).

The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the materials, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Those skilled in the art will recognize how to adapt the features of the exemplified antigenic combinations for immunization against pathogenic Francisella bacteria and related nanolipoprotein particles, composition methods and systems. according to various embodiments and scope of the claims.

All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.

The entire disclosure of each document cited (including webpages patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence. Further, the computer readable form of the sequence listing of the ASCII text file IL-13256-CIP-Sequence-Listing_ST25 is incorporated herein by reference in its entirety.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified may be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the invention and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods may include a large number of optional composition and processing elements and steps.

In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

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1. An antigenic combination comprising an antigenic polysaccharide component from a Francisella bacterium capable of triggering a humoral immune response in an individual, a protein antigen component from the Francisella bacterium capable of triggering a cellular immune response in the individual, and an adjuvant, the antigenic polysaccharide component from the Francisella bacterium, the protein antigen component from the Francisella bacterium, and the adjuvant in a suitable amount to immunize an individual against the Francisella bacterium.
 2. The antigenic combination of claim 1, wherein the antigenic polysaccharide component from the Francisella bacterium is in an amount from about 0.1 to 100 the protein antigen component from the Francisella bacterium is in an amount from about 0.1 to 100 and the adjuvant in an amount from about 0.001 to 200 μg.
 3. The antigenic combination of claim 1, wherein the antigenic polysaccharide component from the Francisella bacterium and the protein antigen component from the Francisella bacterium are at a ratio between 1:0.1 and 1:10 by mass.
 4. The antigenic combination of claim 1, wherein the antigenic polysaccharide component from the Francisella bacterium comprises an LPS molecule and/or a fragment thereof.
 5. The antigenic combination of claim 1, wherein the antigenic polysaccharide component from the Francisella bacterium comprises CPS and/or a derivative thereof.
 6. The antigenic combination of claim 4, wherein the antigenic polysaccharide component from the Francisella bacterium and the protein antigen component from the Francisella bacterium are at a ratio of 1:4 by mass.
 7. The antigenic combination of claim 1, wherein the antigenic polysaccharide has a general formula of (XI):

M1-M4 can each independently be selected from the group consisting of (Xa) to (Xw):

in which L1-L4 can be selected independently from the group consisting of: α (1→2), α (1→3), α (1→4), α (1→5), α (1→6), β (1→2), β (1→3), β (1→4), β (1→5), and β (1→6); and n is 1 to
 1250. 8. The antigenic combination of claim 7, wherein n is 10-200.
 9. The antigenic combination of claim 7, wherein M1, M2, M3 and M4 are each independently selected from the group consisting of α-D-GalNAc (Xm), β-D-QuiNAc (Xs), and β-D-Qui4NFm (Xw).
 10. The antigenic combination of claim 7, wherein M1 and M2 are α-D-GalNAc (Xm).
 11. The antigenic combination of claim 7, wherein the antigenic polysaccharide has a general formula of β-D-Qui4NFm (Xw), α-D-GalNAcAN (Xq), α-D-GalNAcAN (Xq), and β-D-QuiNAc (Xs).
 12. The antigenic combination of claim 7, wherein L1, L2, L3 and L4 are each independently selected from the group consisting of α (1→4), α (1→3), β (1→2), and β (1→4).
 13. The antigenic combination of claim 7, the antigenic polysaccharide further comprises a lipid component.
 14. The antigenic combination of claim 13, wherein the lipid component has a general formula of Y1:

wherein R10 to R13 are each independently represented by hydrogen H or Formula (Y11):

wherein R30 is hydrogen H, OH or represented by Formula (Y12):

R20 and R21 is represented by Formula (Y10):

wherein M10 is hydrogen H or a monosaccharide linked to the oxygen by the corresponding anomeric carbon, the monosaccharide is selected from the group consisting of Formulas (Xa) to (Xw), and wherein Z is a trisaccharide.
 15. The antigenic combination of claim 1, wherein the protein antigen component from the Francisella bacterium comprises one or more protein antigens having SEQ ID NOs: 1-11, 88, and 13-29 and/or one or more protein antigens having at least 30% identity with the Francisella protein antigen having SEQ ID NOs: 1-11, 88, and 13-29.
 16. The antigenic combination of claim 1, wherein the protein antigen component from the Francisella bacterium comprises IglC or a derivative thereof.
 17. The antigenic combination of claim 1, wherein the antigenic polysaccharide component from the Francisella bacterium is from F. tularensis LVS.
 18. The antigenic combination of claim 1, wherein the antigenic polysaccharide component from the Francisella bacterium is from F. tularensis subsp. tularensis.
 19. The antigenic combination of claim 1, wherein the adjuvant comprises at least one of immunostimulatory peptides, muramyl dipeptides, saponins, toxins, oligonucleotide CpG motifs, organic compounds, alpha-galactosyl ceramide, and lipotichoic acid, or hydrophilic adjuvants synthetically appended with a hydrophobic moiety, including microbial derivatives, plant derivatives, and immunostimulatory proteins, immunostimulatory carbohydrates and polysaccharides, and immunostimulatory nucleic acids.
 20. The antigenic combination of claim 1, wherein the adjuvant comprises at least one of AddaVax™, Adju-Phos® adjuvant, Alhydrogel® adjuvant 2%, CFA, IFA, Quil-A® adjuvant, 2′3′-cGAMP VacciGrade™, 3′3′-cGAMP VacciGrade™, c-di-AMP VacciGrade™, 2′3′-c-di-AM(PS)2 (Rp,Rp) VacciGrade™, c-di-GMP VacciGrade™, Chitosan VacciGrade™, CL401 VacciGrade™, CL413 VacciGrade™, CL429 VacciGrade™, Flagellin FliC VacciGrade™Gardiquimod VacciGrade™, Imiquimod VacciGrade™, LPS-EB VacciGrade™, MPLA-SM VacciGrade™, MPLA Synthetic VacciGrade™, N-Glycolyl-MDP VacciGrade™, ODN 1585 VacciGrade™, ODN 1826 VacciGrade™, ODN 2006 Vaccigrade™, ODN 2395 VacciGrade™, Pam3CSK4 VacciGrade™, Poly(I:C) (HMW) VacciGrade™, R848 VacciGrade™, and TDB VacciGrade™.
 21. An immunogenic nanoparticle comprising: one or more scaffold proteins; one or more membrane forming lipids; an antigenic Francisella polysaccharide component comprising, at least one antigenic polysaccharide from Francisella and/or a derivative thereof; a Francisella protein antigen component comprising at least one protein antigen from Francisella and/or a derivative thereof, and an adjuvant, wherein the one or more membrane forming lipids are arranged in a discoidal membrane lipid bilayer stabilized by the one or more scaffold protein and the at least one antigenic polysaccharide from Francisella or the derivative thereof, the at least one protein antigen from Francisella and/or derivative thereof, and the adjuvant are attached to the discoidal membrane lipid bilayer.
 22. The immunogenic nanoparticle of claim 21, further comprising: a functionalized membrane-forming lipid presenting an anchor compound substrate, wherein the at least one protein antigen from Francisella and/or derivative thereof and/or the at least one antigenic polysaccharide from Francisella and/or derivative thereof, is attached to an anchor compound and is further attached to the functionalize membrane-forming lipid through binding of the anchor compound with the anchor compound substrate.
 23. The immunogenic nanoparticle of claim 21, wherein the immunogenic nanoparticle, the adjuvant, the antigenic Francisella polysaccharide component and the Francisella protein antigen component are at a ratio of 1:1:1:1: to 1:50:50:50.
 24. The immunogenic nanoparticle of claim 21, wherein the immunogenic nanoparticle, the adjuvant, the antigenic Francisella polysaccharide component and the Francisella protein antigen component are at a ratio range of 1:2:2:2 to 1:10:10:10.
 25. The immunogenic nanoparticle of claim 21, wherein the immunogenic nanoparticle, the adjuvant, the antigenic Francisella polysaccharide component and the Francisella protein antigen component are at a ratio of immunogenic nanoparticle: antigenic polysaccharide:protein:adjuvant of 1:2:2:2.
 26. The immunogenic nanoparticle of claim 21, wherein the at least one antigenic polysaccharide from Francisella and/or a derivative thereof, comprises at least one of the antigenic polysaccharide from Francisella and/or a derivative having a general formula (XI):

M1-M4 can each independently be selected from the group consisting of (Xa) to (Xw):

in which L1-L4 can be selected independently from the group consisting of: α (1→2), α (1→3), α (1→4), α (1→5), α (1→6), β (1→2), β (1→3), β (1→4), β (1→5), and β (1→6); and n is 1 to
 1250. 27. The immunogenic nanoparticle of claim 21, wherein the at least one protein antigen from Francisella and/or a derivative thereof, comprises at least one protein antigen from Francisella and/or a derivative thereof having SEQ ID NOs: 1-11, 88, and 13-29 and/or one or more protein antigens having at least 30% identity with the Francisella protein antigen having SEQ ID NOs: 1-11, 88, and 13-29.
 28. The immunogenic nanoparticle of claim 21, wherein the adjuvant comprises at least one adjuvant selected from at least one of immunostimulatory peptides, muramyl dipeptides, saponins, toxins, oligonucleotide CpG motifs, organic compounds, alpha-galactosyl ceramide, and lipotichoic acid, or hydrophilic adjuvants synthetically appended with a hydrophobic moiety, including microbial derivatives, plant derivatives, and immunostimulatory proteins, immunostimulatory carbohydrates and polysaccharides, and immunostimulatory nucleic acids.
 29. An immunogenic composition comprising the antigenic combination of claim 1 together with a suitable vehicle, the antigenic combination in an amount effective to induce an immunogenic response against the Francisella bacterium in an individual.
 30. The immunogenic composition of claim 29, wherein components of the antigenic combination are presented on a single same or different carrier.
 31. The immunogenic composition of claim 29, wherein components of the antigenic combination are presented on separate same or different carrier.
 32. The immunogenic composition of claim 30, wherein the carrier is selected from liposomes, polymeric micelles, microspheres, and nanoparticles.
 33. A method of immunizing an individual against an infection of a Francisella bacterium, the method comprising administering to the subject an effect amount of the immunogenic combination of claim 1 alone or within a composition further comprising a suitable vehicle, the antigenic combination in an amount effective to induce an immunogenic response against the Francisella bacterium in an individual.
 34. The method of claim 33, wherein the administering is performed by intranasal inhalation.
 35. The method of claim 33, wherein the administering is performed by intramuscular administration.
 36. A system for immunizing an individual against an infection of a Francisella bacterium, the system comprising an antigenic combination of claim 1, wherein the antigenic polysaccharide component and the protein antigen component are from the Francisella bacterium, and wherein one or more Francisella antigenic polysaccharide and/or derivative thereof comprised in the antigenic Francisella polysaccharide component, one or more protein antigens from Francisella and/or a derivative thereof comprised in the Francisella protein antigen component, and/or one or more adjuvants, are comprised in separate compositions in a form and an amount for simultaneous, combined or sequential use in the method to immunize an individual against the Francisella bacterium.
 37. An immunogenic composition comprising the immunogenic nanoparticle of claim 21 together with a suitable vehicle, the immunogenic NLPs in an amount effective to induce an immunogenic response against the Francisella bacterium in an individual.
 38. A method of immunizing an individual against an infection of a Francisella bacterium, the method comprising administering to the subject an effective amount of the immunogenic nanolipoprotein particle of claim 21 alone or within a composition further comprising a suitable vehicle, the immunogenic nanolipoprotein in an amount effective to induce an immunogenic response against the Francisella bacterium in an individual.
 39. The method of claim 38, wherein the administering is performed by intranasal inhalation.
 40. The method of claim 38, wherein the administering is performed by intramuscular administration.
 41. The antigenic combination of claim 1, wherein the protein antigen component from the Francisella bacterium comprises a peptide selected from SEQ ID NO: 33, 37, 38, 45, 46, 67, 68, 72 and
 73. 42. The antigenic combination of claim 1, wherein the protein antigen component from the Francisella bacterium comprises a protein peptide selected from SEQ ID NO: 33, 38, 45, 67, and
 43. The antigenic combination of claim 1, wherein the protein antigen component from the Francisella bacterium comprises a peptide having SEQ ID NO:
 33. 44. The antigenic combination of claim 1, wherein the protein antigen component from the Francisella bacterium comprises a peptide having SEQ ID NO:
 81. 45. The immunogenic nanoparticle of claim 21, wherein the Francisella protein antigen component comprises a protein peptide selected from SEQ ID NO: 33, 37, 38, 45, 46, 67, 68, 72 and
 73. 46. The immunogenic nanoparticle of claim 21, wherein the Francisella protein antigen component comprises a protein peptide selected from SEQ ID NO: 33, 38, 45, 67, and
 73. 47. The immunogenic nanoparticle of claim 21, wherein the Francisella protein antigen component comprises a protein peptide having SEQ ID NO:
 33. 48. The immunogenic nanoparticle of claim 21, wherein the Francisella protein antigen component comprises a protein peptide having SEQ ID NO:
 81. 